Mitosis, during which duplicated chromosomes are segregated to opposite poles of the cell must occur accurately to ensure that daughter cells contain an intact copy of the genome. Defects in this process can lead to changes in chromosome number, a driving force behind cancer formation. Sororin plays a key role in maintaining sister chromatid cohesion by interacting with the cohesin complex. The regulation of Sororin is poorly understood. We have created a Sororin mutant in which nine putative Cdk1 phosphorylation sites have been converted to alanine ("Sororin9A"). Sororin9A remains constitutively associated with chromosomes and increases cohesion between sister chromatids during mitosis. We propose to analyze the mechanism by which Cdk1-mediated phosphorylation regulates the function of Sororin in sister chromatid cohesion. This proposal entails two specific aims.
Aim 1. Regulation of Sororin phosphorylation by Cdk1 and PP2A. The relative efficiency of phosphorylation at each of nine putative Cdk1 sites in Sororin is not known. Phospho-tryptic mapping will be used to determine which of the nine Cdk1 sites are major phosphorylation sites in vivo. With this information, we will embark on a more directed analysis of the role of specific phosphorylation sites in Sororin function. Phosphorylation of Sororin is predicted to inhibit its ability to stabilize sister chromatid cohesion. We plan to test this idea directly by analyzing the cellular effects of Sororin mutants in which individual serines/threonines have been converted to glutamic acid to mimic the constitutively phosphorylated state. Finally, PP2A appears to dephosphorylate Sororin during mitotic exit. The regulation of Sororin at the inner centromere by PP2A at the kinetochore will be determined.
Aim 2. Role of Sororin phosphorylation in sister chromatid cohesion. Sororin9A increases sister chromatid cohesion, an effect that is hypothesized to be due to the persistence of the cohesin complex on chromosome arms. Immunofluorescence will be used to measure the amount of cohesin subunits associated with chromosomes after overexpression of Sororin9A. Studies in Xenopus extracts suggest that the effect of Sororin phosphorylation is due to changes in the association of Sororin with the cohesin accessory factor Pds5. We propose to determine the effect of mutating the Pds5-docking site on Sororin, a region that contains the essential motif "FGF". Mutating this motif to AGA in Sororin9A should block the increase in cohesion caused by Sororin9A. The current model for the role of Sororin in sister chromatid cohesion suggests that it competes with Wapl for binding to Pds5. Binding of Wapl to Pds5 releases the cohesin ring along chromosome arms during prophase, while Sororin may antagonize this effect. We predict that overexpressing Wapl will reverse the effects of Sororin9A on cohesion. Determining the role of Sororin phosphorylation on sister chromatid cohesion will fill an important gap in our understanding of this essential process.
Project Narrative More than half a million people in the US die every year due to cancer, a disease characterized by uncontrolled cell division, and inaccurate segregation of chromosomes. The protein Sororin plays an essential role in cell division and understanding how it is regulated will provide important insight into how human cancer cells divide and possibly how to kill them. Increased proliferation of fibroblasts and smooth muscle cells has also been implicated in diseases of the cardiovascular system, and knowing how Sororin is regulated may also provide insight into this spectrum of diseases.