Publications by authors named "Darya Goldman"

2 Publications

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Synthetic-Evolution Reveals Narrow Paths to Regulation of the Mitotic Kinesin-5 Cin8.

Int J Biol Sci 2019 2;15(6):1125-1138. Epub 2019 May 2.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in . This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.
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http://dx.doi.org/10.7150/ijbs.30543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567808PMC
April 2020

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase.

Cell Mol Life Sci 2017 09 28;74(18):3395-3412. Epub 2017 Apr 28.

Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, PO Box 653, 84105, Beer-Sheva, Israel.

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.
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http://dx.doi.org/10.1007/s00018-017-2523-zDOI Listing
September 2017