Abstract
The condensin complex plays a key role in organizing mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. In this study, we dissect the role of a putative Cdk1 site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. We examined the functional significance of this phosphorylation mark by mutating the orthologous site of CAP-D3 (CAP-D3(T1403A)) in chicken DT40 cells. We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal INCENP localization. Our results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes. We present the first in vivo example that alters the ratio of condensin I:II on mitotic chromosomes. Our results demonstrate this ratio is a critical determinant in shaping mitotic chromosomes.
Highlights
A conserved Cdk1 site in CAP-D3 activates condensin II
We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal Inner centromere protein (INCENP) localization
A streptavidin-binding peptide (SBP) GFP tag was engineered into the chicken CAP-D3 DNA at the C terminus for localization and purification purposes [33]
Summary
A conserved Cdk site in CAP-D3 activates condensin II. Results: Mutation of CAP-D3 Thr-1403 in chicken disrupts prophase and leads to chromosome hypercondensation. We dissect the role of a putative Cdk site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. A recent study has provided the important link between Cdk phosphorylation and activation of condensin II in mitosis [29] It identified a Cdk site at threonine 1415 in the human CAPD3/condensin II subunit as being required to activate the complex during prophase, and allow Plk to be recruited along the chromosome axis. Our data suggest that Thr-1403 is a key site in setting the balance of condensin I and II on mitotic chromosomes
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