Faculty and Research
Faculty by Name
Michael Rape
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Michael Rape
Assistant Professor of Cell and Developmental Biology*
*And Affiliate, Division of Biochemistry and Molecular Biology.
Research Interests
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Ubiquitination is a key regulator of proliferation and differentiation in all eukaryotes. It is carried out by a cascade of three different classes of enzymes, E1, E2, and E3. In humans alone, there are ~40 E2s and more than 600 E3s, making ubiquitination enzymes one of the most abundant and diverse family of enzymes. We know the substrates of only few of these enzymes.
We do know that ubiquitination is essential for proliferation and differentiation. The loss of the E3 anaphase-promoting complex APC/C, for example, arrests cells in mitosis, and blocks their ability to segregate chromosomes. The E3 SCF with its many substrate-targeting F-box proteins drives many essential ubiquitination reactions throughout the cell cycle, and its loss cannot be tolerated by dividing cells. Inhibiting these enzymes is an intriguing strategy of blocking the unwanted proliferation of cancer cells.
The importance of ubiquitination for cell cycle control is underscored by the tight links between deregulated ubiquitination and tumorigenesis. Loosing the activity of the E3 Brca1/Bard1, for example, can cause breast and ovarian cancer; the overexpression of the ubiquitin ligase Mdm2, which targets the tumor suppressor p53 for degradation, is observed multiple types of cancers; and mutations that interfere with the recognition of b-catenin by its ubiquitin ligase are responsible for most cases of colon cancer. Again, small molecules that would correct the activities of rampant ubiquitination enzymes would be exciting additions to the currently available set of chemotherapeutics.
Our ability to exploit the ubiquitin system as a target for drug discovery has been hampered by our limited knowledge of essential enzymes and their substrates, and particularly, by our lack of understanding mechanisms of ubiquitination.
Therefore, we are interested in:
Discovering ubiquitination enzymes controlling proliferation and differentiation
Identifying the pathways regulated by these enzymes during cell cycle control
Dissecting the biochemical mechanisms of ubiquitination
Isolating small molecule agonists and antagonists of ubiquitination
Current Projects
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Identification of regulators of proliferation and differentiation
We are using complementary strategies to isolate ubiquitination enzymes that regulate proliferation and differentiation. For example, we are performing high-content, imaging based siRNA-screens to identify E3s and deubiquitinating enzymes important for cell cycle progression and differentiation of embryonic stem cells, or for tissue-specific networks of proliferation. We are also purifying ubiquitination enzymes from cell extracts or stable cell lines, and subject them to mass spectrometry. Both strategies have been highly successful and led to the isolation of novel enzymes required for cell cycle control. For example, we recently discovered a novel enzyme overexpressed in breast cancer, the function of which is to regulate the mitotic spindle.
Identification of cell cycle pathways regulated by ubiquitination
After having discovered ubiquitination enzymes that regulate proliferation and differentiation, we proceed by identifying the cellular pathways controlled by these enzymes. This can involve cell biological approaches, such as depleting the enzyme in question by siRNA and studying effects on the cell cycle by immunofluorescence or live cell imaging. In addition, it often entails the characterization of binding partners or regulators through immunoprecipitation and mass spectrometry. The most direct yet also most difficult approach to understanding the role of novel enzymes in cell cycle control is to identify their substrates. To this end, we have spearheaded in vitro expression cloning strategies that have allowed us to isolate several novel substrates of the ubiquitin ligase anaphase-promoting complex (APC/C).
Dissection of mechanisms of ubiquitin chain formation
Substrates can be modified with one ubiquitin moiety or with ubiquitin chains. Those ubiquitin chains can be extraordinarily different in structure, allowing them to encode different information - the consequences of ubiquitination range from activation of proteins to their degradation. Surprisingly, the mechanisms of ubiquitin chain formation are poorly understood. We have developed systems to study substrate ubiquitination in vitro. This enables us to determine the structure of ubiquitin chains, dissect their mechanism of assembly, and identify the rate-limiting step of the reaction. For example, we have recently shown that the human APC/C assembles a novel type of ubiquitin chain, which is linked through lysine 11. We then isolated specificity factors for this reaction, for example the rate-limiting E2 of the APC/C, UbcH10. Both discoveries may provide novel avenues into inhibiting this essential cell cycle regulator as a means of cancer treatment.
Isolation of small molecule agonists and antagonists of ubiquitination
Because ubiquitination is essential for proliferation and differentiation in all eukaryotes and responsible enzymes are often misregulated in cancer, targeting ubiquitination enzymes by small molecules has great therapeutic potential. Accordingly, an inhibitor of the 26S proteasome has recently been approved by the FDA for the treatment of multiple myeloma. We are currently setting up miniaturized assays that allow us to screen for small molecule agonists or antagonists of E2 enzymes, which are easy to purify and assay in vitro.
Selected Publications
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Jin, L.*, Williamson, A.*, Banerjee, S., Phillip,
I. , and Rape M. (2008). Mechanism of ubiquitin chain formation by the human Anaphase-Promoting Complex. Cell 133, 653-665.Reddy, S.K.*, Rape, M.*, and Kirschner M.W. (2007). Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921-925.
Stegmeier, F.*, Rape, M.*, et al. (2007). Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446, 876-881.
Rape, M., Reddy, S.K. and Kirschner, M.W. (2006). The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124, 89-103.
Richly, H.*, Rape, M.*, Braun, S., Rumpf, S., Hoege, C., and Jentsch S. (2005). A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 120, 73-84.
Rape, M. and Kirschner, M.W. (2004). Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature 432, 588-595.
Rape, M., Hoppe, T., Gorr,
I. , Kalocay, M., Richly, H., and Jentsch S. (2001). Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48. Cell 107, 667-677.Song, L., and Rape M. (2008). Reverse the curse: cell cycle regulation by deubiquitination. Curr. Opin. Cell. Biol. 20, 156-63.
* These authors contributed equally to this work.
Last Updated 2009-06-24