Cell Cycle Control Group
Group Leader: Professor Hiro Yamano
- Maggie Hanwell, Research Assistant
- Michelle Trickey, Postdoc
- Helene Labit, Postdoc
- Kazu Fujimitsu, Postdoc
- Hiroko Yaguchi, Postdoc
Our bodies are composed of many millions of individual cells and each cell inherits genetic information through ordered cell cycle events including DNA replication and chromosome (DNA) segregation. Cancer is a disease of cells causing uncontrolled cell division. Therefore, a full understanding of the mechanisms underlying cell cycle control is required for the fight against cancer.
We are studying how cell growth and the cell cycle are controlled by ubiquitin-mediated proteolysis. We are in particular focusing on a key enzyme called the anaphase-promoting complex/cyclosome (APC/C), which plays an essential role in dividing and non-dividing cells.
The Ubiquitin-Proteasome System (UPS) in eukaryotic cells uses a small molecule called ubiquitin for protein tagging (ubiquitylation) and this acts as a signal for protein destruction. The UPS is essential for degrading damaged and misfolded proteins and also for the degradation of short-lived regulatory proteins during processes such as the cell cycle, transcription, signal transduction, immuno-responses and development.
The anaphase-promoting complex/cyclosome (APC/C) is one of the major cellular ubiquitin ligases, that ensures accurate cell cycle progression and faithful chromosome segregation to daughter cells. Aberrations in chromosome segregation can lead to aneuploidy with irregular numbers of chromosomes and birth defects such as Down's syndrome, which could play a significant role in tumorigenesis. There is also increasing evidence that the APC/C plays an important role in terminally differentiated cells and non-dividing cells such as brain.
Hence, the APC/C is a key enzyme in cells and its study should help us to understand the cell cycle as well as cell signaling and cell differentiation. The APC/C system could be a key drug target for control and monitoring of cell cycle progression.
The APC/C consists of at least 11 subunits (1.5 MDa complex) and thus behaves as a mega-enzyme or cellular nano-machine. Whilst most of the subunits of the APC/C have been identified, very little is understood about their precise regulation and mechanisms of ubiquitylation. We have established a cell-free system based on Xenopus egg extracts that recapitulates several aspects of the APC/C-dependent ubiquitylation and proteolysis.
We seek to decipher the mechanisms by which the APC/C recognises substrates and ubiquitylates them, and to unravel how the APC/C activities are controlled by the regulatory networks such as checkpoints, kinases and phosphatases. In order to study the in vivo function and regulation of the APC/C, we use the fission yeast, Schizosaccharomyces pombe as a model organism, which is highly amenable to genetic, biochemical and genomic studies. These combined approaches greatly improve our understanding of the molecular mechanisms behind the ubiquitin system and how it works to target and degrade proteins in the cell.
Furthermore, our work will contribute to more effective strategies for developing drugs that will not only be useful in killing cancer cells but also aid the diagnosis and treatment of cancer and other diseases.
Eguren, M, Garcia, F, Lopez-Contreras, AJ, Fujimitsu, K, Alvarez-Fernandez, M, Yaguchi, H, Luque-Garcia, JL, Fernandez-Capetillo, O., Muñoz, J, Yamano, H and Malumbres, M. (2014) A new synthetic lethal interaction between APC/C and topoisomerase poisons uncovered by proteomic screens. Cell Reports, 6, 1-14.Pubmed
Trickey, M, Fujimitsu, K. and Yamano, H. (2013) APC/C-mediated proteolysis of Ams2 in G1 ensures the coupling of histone gene expression to DNA replication in fission yeast. J. Biol. Chem., 288: 928-937. Pubmed
Labit, H., Fujimitsu, K., Bayin, N.S., Takaki, T., Gannon, J. and Yamano, H. (2012) Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C ubiquitin ligase. EMBO J., 31:3351-3362. Pubmed
Kimata, Y., Kitamura, K., Fenner, N. and Yamano, H. (2011) Mes1 controls the meiosis I to meiosis II transition by distinctly regulating APC/C co-activators Fzr1/Mfr1 and Slp1 in fission yeast. Mol. Biol. Cell, 22: 1486-94. Pubmed
Manchado, E., Guillamot, M., de Cárcer, G., Eguren, M.,Trickey, M., García-Higuera, I., Moreno, S., Yamano, H., Cañamero, M., and Malumbres, M. (2010) Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl and the PP2A/B55 α,δ phosphatase. Cancer Cell, 18: 641-654. Pubmed
Takayama, Y., Mamnun, Y.M., Trickey, M., Dhut, S., Masuda, F., Yamano, H., Toda, T. and Saitoh, S. (2010) Hsk1- and SCFpof3-dependent proteolysis of S. pombe Ams2 ensures histone homeostasis and centromere function. Dev. Cell, 18: 385-396. Pubmed
Ors, A., Grimaldi, M., Kimata, Y., Wilkinson, C.R.M., Jones, N. and Yamano, H. (2009) The transcription factor Atf1 binds and activates the APC/C ubiquitin ligase in fission yeast. J. Biol. Chem., 284: 23989-23994. Pubmed
Yamano, H., Trickey, M., Grimaldi, M. and Kimata, Y. (2009) In vitro assays for the anaphase-promoting complex/cyclosome (APC/C) in Xenopus egg extracts. Methods Mol. Biol., 545: 287-300. Pubmed
Trickey, M., Grimaldi, M. and Yamano, H. (2008) The anaphase-promoting complex/cyclosome (APC/C) controls repair and recombination by ubiquitylating Rhp54 in fission yeast. Mol. Cell. Biol., 28: 3905-3916. Pubmed
Kimata, Y., Trickey, M., Izawa, D., Gannon, J., Yamamoto, M. and Yamano, H. (2008) A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast. Dev. Cell, 14: 446-454. Pubmed
Fry, A.M. and Yamano, H. (2008) Under arrest in mitosis: Cdc20 dies twice. Nat. Cell Biol., 10: 1385-1387. Pubmed
Kimata, Y., Baxter, J.E., Fry, A.M. and Yamano, H. (2008) A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment. Mol. Cell, 32: 576-583. Pubmed
Fig 1 The cell cycle is an elaborate series of events by which cells duplicate their chromosomal DNA and segregate the chromosomes into two daughter cells. A complex network of proteins regulates the events and transitions during the cell cycle. In particular, three elements, Cdks/cyclins, checkpoints and ubiquitin-proteasome system (UPS) play major roles. The deregulation causes genomic instability and tumorigenesis.
Fig 2 Ubiquitin-mediated proteolysis. Ubiquitin serves as a molecular tag that marks proteins for destruction. Ubiquitin is first attached by a thioester bond to an ubiquitin-activating enzyme (E1) in an ATP-dependent manner, then transferred to an ubiquitin-conjugating enzyme (E2), and finally the ubiquitin is conjugated onto the target substrates either by E2 alone or, more often, in conjugation with a substrate-specific ubiquitin ligase (E3). Proteins carrying long chains or trees of ubiquitin are delivered to the 26S proteasome for degradation.
Fig 3 A key regulator of the cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase (E3).
Fig 4 Purification of the soluble APC/C from Xenopus egg (Xl) and HeLa cell (Hs) extracts.
Fig 5 We use a combination of biochemistry, genetics and cell biology. These complementary approaches provide a more powerful way to study biological events at the molecular level.