SUMOylation: Wrestling with the Regulation of APC/C

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 column through column chromatography. 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.

11 Comments

  1. Hey Calvin, great review! It is always really interesting to see what kinds of mechanisms are responsible for, as you said, regulating the master regulator. One question I have is that, given that APC/C has fifteen subunits, do we know much about regulation at other subunits on the protein? With there being fifteen of them it seems likely that regulation is not just occurring on subunit 4.

    1. Hey Brandon, thank you for the great question. After looking at some literature, it appears that APC/C has over 42 different phosphorylation spots with many of them relating to mitosis! The literature I saw did not specify which subunits have what phosphorylation but I would feel confident to say that not all 42 phosphorylation sites are on the same subunit. I would imagine that the phosphorylation sites are mainly on the few subunits that have specific jobs.

  2. Hi Calvin, this looks like a very thorough study done on one mechanism of SUMO control in the cell. I was wondering if you could elaborate more on what the significance of the findings are. Given that the authors have determined which residues get SUMOylated, does this open up the possibility for a drug target or specific alteration of either the SUMO mechanism or APC/C interaction in the context of cancer treatment.

    1. Hey Suzi, thanks for the important question. You are exactly right about the work being applied for cancer treatment. The authors stated that knowing how APC/C is regulated through SUMOylation is a key step before it can become druggable. I would imagine, a drug company could control whether APC/C is turned on or off by SUMOylation thus controlling whether the cell divides or now. I could easily see the drug companies come up with a small organic molecule that binds to APC4 to prevent SUMOylation occurring.

  3. Hi Calvin, good work reviewing this paper with an interesting translational potential. One of the main punchlines was that SUMOylation of APC/C results in more ubiquitinylation of KIF18B, which allows the cell to progress into anaphase. You mentioned towards the end that this knowledge can allow for a new druggable target to treat cancer. Do you think it is possible to drug the specific SUMO that binds APC/C to slow down cell cycle progression in cancer?

    1. Hey Brandon, thank you for the great question. I think it would be really hard to selectively target the SUMO binding to APC/C since there is not an APC/C specific SUMO but I do believe it could be possible to drug the APC4 subunit that handles the SUMOylation. With this being said, it would be tricky to insure that only cancer cells are impacted by the drug since the SUMOylation machinery is present in more than just cancer cells.

  4. Hi Calvin,
    Thanks for the interesting review! This has nice ties to what we’ve learned in class about SUMOylation. At one point in the paper, the authors note that the lysine residues in human APC4 are not conserved in yeast APC4. I’m curious as to why they decide to do research into yeast. Are they simply noting that this is something conserved only in eukaryotes? And why might this be relevant?

  5. Hi Calvin! This was a very interesting review with a strong connection to the material in Unit 3. It would be interesting to see any further work on the sumoylation of APC/C to eventually develop drugs that target the sumo protein and its implication of cancer cell regulatory processes. I do have a question on the authors’ results on mitotic phases when under knock out of the sumoylating enzymes. You mention that the authors witnessed an increased rate of chromatin bridging, but a slower mitotic rate. I’m no expert in cell division, but is this finding this contradictory?

  6. Hi Calvin,

    Thank you for your review, it was really helpful. You mentioned towards the end that the study has translational potential to treat cancer. However, considering that SUMOylation has so many downstream consequences that often cannot be predictable, isn’t it reasonable to believe that targeting the sumo protein would have detrimental effects on other systems and regulation in the body as well? Do you think this kind of therapy would be “cancer specific” and be able to combat cancer while not impairing other functionalities in the body?

  7. Thanks for the review and the amusing title Calvin! SUMOylation is extremely relevant to our course so I am glad I got to learn a bit more about it in the context of a possible drug target. I just got a little confused when you discussed the part regarding when the authors forced the HeLa cells to express His-tagged SUMO2 and pulling the subunit off of a nickel. I am a bit confused how exactly this allowed the authors to know that APC4 subunit was necessary for SUMOylation. Do you have any more insight into this?

  8. Great read Calvin. The effects of PTMs of the cellular environment is incredibly complex and intricate, and as the authors found out: very hard to map and understand. These complex interactions only follow the complex regulatory interactions between proteins and genes, especially in the context of cancer. Is SUMOylation responsible for normal function of these oncoproteins as well as the cancer causing dysregulations? If SUMOylated proteins are the risk factor, do we understand the mechanism by which these are added to the protein?

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