SUMOylation: Wrestling with the Regulation of APC/C

Author: Calvin Inners

The paper can be accessed here: https://www.nature.com/articles/s41467-018-03486-4

 

The image above depicts the SUMOylation and ubiquitination of ACP/C through its APC4 domain to add ubiquitins to KIF18B to ensure an error-free mitotic progression through anaphase.

Cancer has been one of the deadliest disease in the history of mankind. With almost six-hundred thousand deaths in 2016, the pressure to understand the mechanism of the disease works and how it is caused remains a priority (Siegel, Miller, and Jemal 2016). In recent history, a lot of work has been done relating the dysregulation of proteins to the oncogenesis of cancer. The dysregulation of protein can lead to dysfunctions such as improper cell cycle progression, which can in turn create cancer causing problem such as aneuploidy (Holland and Cleveland 2012). To understand how these proteins are dysregulated, we first need to know how they are regulated.

One important step to knowing how proteins are regulated is to look at protein posttranslational modifications (PTM) such as phosphorylation and ubiquitylation. These modifications have become well known for their countless roles including cell cycle regulation. Anaphase-promoting complex/cyclosome (APC/C) is a protein complex with fifteen subunits. Additionally, APC/C is an E3 ubiquitin ligase that has a major role in degrading proteins that withhold the cell cycle such as securin (Peters 2006). Securin is a highly regulated protein since it inhibits Cohesion which is responsible for cleaving the chromatid holding protein, Seperase. These three proteins are vital for a mistake-free anaphase necessary to prevent aneuploidy and cancer (Wang et al. 2003). Therefore, APC/C regulates these proteins. With this being said, it is important to understand what regulates the master regulator. While we known a considerable amount about phosphorylation and ubiquitylation we do not know a lot about ubiquitylation’s fun sounding cousin, small ubiquitin-like modifier (SUMO) (Schimmel et al. 2014). Since we do not know how SUMOylation regulates APC/C, the Eifler et al. group decided to investigate the role SUMOylation plays regarding APC/C to further elucidate its role in aneuploidy and carcinogenesis.

Eifler et al. started their paper off with determining whether the SUMOylation of APC/C impacts the mitotic index (how rapidly a cell divides) of the HeLa cells. After knocking out the two enzymes responsible for SUMOylation of APC/C, SUMO-activating enzyme 1 and SUMO-activating enzyme 2, separately, the authors saw an increase in the time it took the cells to go from metaphase to anaphase in both knockouts with a more prominent time increase in the SUMO-activating enzyme 2 knockout. Additionally, the authors looked at chromatin bridges (when the telomers of sister chromatids are stuck together) due to these knockouts. The chromatin bridges formed at a much higher rate when the knockout occurred allowing the authors to know why it took more time to progress from metaphase to anaphase. Once it was determined that the dysregulation of APC/C can cause chromatin bridges, the authors needed to determine where on the protein complex was it regulated.

APC/C has been reported to have fifteen subunits. It is important to know which of these subunits is responsible for the SUMOylation. Eifler et al. used bioinformatic tools to see that similar proteins had a lysine acceptor site but the corresponding similar protein in APC/C did not have the lysine acceptor site. Since this did not lead to finding the right subunit, the group forced the HeLa cells to express His-tagged SUMO2 which bound to the APC4 subunit of APC/C. This allowed the authors to pull the SUMOlated APC4 subunit off a nickel. This allowed the authors to know that the APC4 subunit of APC/C was the subunit necessary for SUMOylation.

Once it was known that APC4 was needed for SUMOylation, the authors investigated where the SUMOylation occurred on the subunit. Through mutating two lysine residues to arginine in the 772 and 798 site the authors were able to see that APC/C was not longer able to be SUMOylated on the APC4 subunit. Once it was determined that these residues were responsible for SUMOylation, the authors took it a step further and to determine if the SUMOylationg and phosphorylation events of APC4 were connected. To do this the authors mutated two downstream serines (S777 and S779) to alanines and saw that if phosphorylation was inhibited then the SUMOylation was inhibited as well. Once this was found out, the Eifiler et al. group decided to tease apart the role SUMOylation has on mitotic progression.

To determine the role SUMOylation has on mitotic progression, the authors knocked down the APC4 subunit and saw an increased time to go from metaphase to anaphase suggesting that SUMOylation has a significant role in regulating the master regulator. To explore the effects of SUMOylation on APC4 Eifler et al. purified SUMOylated APC/C to see what was attached to the protein complex. When this was done, they noticed the APC/C substrate Hsl1 and coactivator CDH1 was attached. When the group monitored the ubiquitylation of Hsl1 they noticed that the majority of the ubiquitylation was done by the SUMOylated APC4.

After analyzing the ubiquitylation of Hsl1, the group used mass spectrometric data to analyze which kinesins (proteins responsible for controlling the length of the microtubule filaments attached to the kinetochore of sister chromatids) were upregulated and saw KIF18B was upregulated. This indicated that SUMOylated APC4 preferentially regulated KIF18B. The Eifler et al. group finished their paper by determining that KIF8B was ubiquitylated after the SUMOylation of APC4. They determined this by looking at SUMO interaction motifs through various mutation studies and assays.

Through various assays and mutation studies, the Eifler et al. group was highly successful at teasing apart the unknown role SUMOylation of ACP/C has on cell cycle progression. As seen in class, we know that these processes are highly complex and often overlap with other regulation mechanisms. Additionally, this paper elucidates the importance of regulation through SUMOylation.

Through this study of SUMOylation, the authors were able to understand another regulatory failure that could occur promote oncogenesis. This new knowledge will allow for a new druggable target to treat cancer. While this is a significant leap forward, it is sill important to keep in mind that there is a lot of work that still needs to be done. The paper is one example of the complex regulatory mechanism that occurs to perform seemingly simple cell functions, but it is still important to continue to look for other regulatory functions of PTM and other alterations of proteins.

 

References

Holland, Andrew J., and Don W. Cleveland. 2012. “Losing Balance: The Origin and Impact of Aneuploidy in Cancer: ‘Exploring Aneuploidy: The Significance of Chromosomal Imbalance’ Review Series.” EMBO Reports 13 (6): 501–14. https://doi.org/10.1038/embor.2012.55.

Peters, Jan-Michael. 2006. “The Anaphase Promoting Complex/Cyclosome: A Machine Designed to Destroy.” Nature Reviews Molecular Cell Biology 7 (9): 644–56. https://doi.org/10.1038/nrm1988.

Schimmel, Joost, Karolin Eifler, Jón Otti Sigurðsson, Sabine A. G. Cuijpers, Ivo A. Hendriks, Matty Verlaan-de Vries, Christian D. Kelstrup, et al. 2014. “Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein.” Molecular Cell 53 (6): 1053–66. https://doi.org/10.1016/j.molcel.2014.02.001.

Siegel, Rebecca L., Kimberly D. Miller, and Ahmedin Jemal. 2016. “Cancer Statistics, 2016.” CA: A Cancer Journal for Clinicians 66 (1): 7–30. https://doi.org/10.3322/caac.21332.

Wang, Qing, Caroline Moyret-Lalle, Florence Couzon, Christine Surbiguet-Clippe, Jean-Christophe Saurin, Thierry Lorca, Claudine Navarro, and Alain Puisieux. 2003. “Alterations of Anaphase-Promoting Complex Genes in Human Colon Cancer Cells.” Oncogene 22 (10): 1486–90. https://doi.org/10.1038/sj.onc.1206224.

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