The role of Cyclin A in terms of sister chromatid cohesion

Intrigued by the observation that Cyclin A is required for SCS in meiosis II of mouse oocytes we asked for the effect induced by overexpression of non-degradable Cyclin A in mitotic cells.

Indeed, non-physiological levels of Cyclin A or its presence in late mitosis cause a pronounced cohesion defect as determined by chromosome spreading using cells arrested in prometaphase. Several studies suggested that the cohesion protection mechanism at the centromere is abrogated by Sgo2 relocation when bipolar tension is applied across sister kinetochores in metaphase II (Gomez et al., 2007; Lee et al., 2008). The authors only addressed the localization of Sgo2 by immunostaining and assumed that PP2A would redistribute accordingly since depletion of Sgo2 leads to a complete loss of centromeric PP2A. However, in immunofluorescence experiments conducted by Chambon et al. Sgo2 indeed relocalized upon bipolar tension but the signal of PP2A and centromeric Rec8 remained overlapping (Chambon et al., 2013). This observation suggests that Sgo2 might be only responsible for the initial recruitment of PP2A to the centromere but afterwards the localization of both proteins is independent of each other. Furthermore, Chambon and colleagues showed that SCS in oocytes depends on the PP2A inhibitor I2PP2A, which co-localizes with the PP2A enzyme to centromeres at metaphase II. Interestingly, experiments studying PP2A in a different pathway

Discussion

in meiosis II to boost I2PP2A’s inhibition capacity by phosphorylation. However, we were not able to gain evidence for an involvement of I2PP2A in the cohesion defect caused by Cyclin A overexpression in mitotic cells. Neither did I2PP2A overexpression alone cause any premature SCS nor did co-overexpression of I2PP2A and Cyclin A increase the cohesion defect compared to Cyclin A expression alone (data not shown). In addition, we were unable to detect I2PP2A at the centromere of prometaphase cells although non-degradable Cyclin A was expressed (data not shown). Moreover, a siRNA mediated depletion of I2PP2A did not rescue the Cyclin A induced cohesion defect (data not shown). This result, however, is inconclusive since we were not able to verify the knockdown efficiency due to lack of a functional antibody against I2PP2A.

Remarkably, there are even more examples for PP2A inhibitors that are regulated by phosphorylation. For example, it is well established that PP2A-B55 is regulated by the protein Ensa which has to be phosphorylated by the kinase Gwl to become inhibitory (Mochida et al., 2010). Most importantly, a very recent study described a protein, namely Bod1, that is required for the fine tuning of PP2A-B56 activity at the kinetochore. The chromosome alignment defect observed upon Bod1 depletion is not rescued by expression of a Bod1 variant in which a potential Cdk1 phosphorylation site was exchanged to alanine (Porter et al., 2013).

These data suggest that inhibition of PP2A may depend on phosphorylation of Bod1 by Cdk1.

The sister chromatid cohesion failures caused by Cyclin A overexpression could be attributed to hyperphosphorylation and, thus, increased activity of Bod1. A too tight inhibition of PP2A might render centromeric cohesin susceptible to Wapl activity. It will be very interesting to test the effect of Bod1 depletion or overexpression on the cohesion defect induced by non-physiological levels of Cyclin A.

Nonetheless, it is also possible that Cyclin A-Cdk inhibits PP2A by direct phosphorylation. Very recent data strongly suggest that direct phosphorylation of the phosphatase is involved in its regulation (Grallert et al., 2015). To test this, one could simply monitor the phosphatase activity of PP2A with and without pre-incubation with Cyclin A-Cdk. PP2A can be obtained by purification of the tagged and overexpressed B56 subunit, which will co-precipitate subunits A and C. Cyclin A-Cdk can be isolated as already described in this study and a standard, commercially available malachite green assay can be used to quantify phosphatase activity.

We speculated that Cyclin A might be involved in the regulation of prophase pathway signaling. Therefore, we asked whether Cyclin A-Cdk is able to phosphorylate Sororin.

Discussion

Intriguingly, Cyclin A-Cdk and Cyclin B-Cdk modified Sororin to a similar extent. An interesting idea is that both Cyclins have to phosphorylate Sororin before it can be displaced from cohesin by Wapl. A straightforward experiment can help to elucidate if Cyclin A-Cdk and Cyclin B-Cdk attach phosphate groups to different residues of Sororin. Sororin has to be incubated with Cyclin B-Cdk in the presence of cold ATP. The pre-phosphorylated Sororin is subsequently incubated with Cyclin A-Cdk in the presence of radioactively labelled ATP and subjected to autoradiography. One sample with addition of new Cyclin B-Cdk serves as control. A signal in the autoradiography after incubation with Cyclin Cdk would indicate that Cyclin A-dependent Cdk can phosphorylate sites that cannot be targeted by Cyclin B-Cdk.

Another kinase, which might be interesting in terms of prophase pathway control is Nek2A.

Similar to Cyclin A this kinase is degraded in early mitosis in an APC/C-dependent manner (Hames et al., 2001; Hayes et al., 2006). An easy and straightforward experiment is to overexpress a non-degradable variant of Nek2A and test for cohesion defects in metaphase arrested cells. Furthermore, it could be worthwhile to perform in vitro kinase assays to test if Sororin is a substrate of Nek2A. A positive result could point to an involvement of Nek2A in cohesin ring opening. An additional thrilling in vivo experiment would be to deplete Cyclin A and/or Nek2A in cells and subject the cells to chromosome spreading. An enrichment of mitotic chromosomes without resolved arm cohesion compared to control cells would suggest a defect in prophase pathway signaling upon depletion of the corresponding kinase(s).

For quite some time the biological purpose of the prophase pathway was not clear. However, very recently Rowland and coworkers could show that resolution of arm cohesion in prophase ensures correct decatenation of sister chromatids. When they shut down prophase pathway activity by Wapl depletion they observed failures during chromosome segregation such as lagging chromosomes (Haarhuis et al., 2013). Since it is important for genome integrity a comprehensive understanding of prophase pathway signaling is of great interest. In this work we provide hints that Cyclin A might be involved in the control of this vital cellular process and, hence, has the potential to shape future prophase pathway research.

Material and Methods

4. Material and Methods