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Michael P. Rape

Michael Rape

Howard Hughes Investigator and Professor of Cell and Developmental Biology*
*And Affiliate, Division of Biochemistry and Molecular Biology.

Lab Homepage: http://mcb.berkeley.edu/labs/rape/

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Research Interests

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 38 E2s and more than 600 E3s, making ubiquitination enzymes one of the most abundant and diverse family of enzymes. Loss of essential ubiquitination enzymes can lead to rapid cell cycle arrest.

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. 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 that control 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

1. Dissecting the ubiquitin code

 

Ubiquitin is covalently linked to lysine residues in substrate proteins. The transfer of a single ubiquitin moiety (monoubiquitination) usually results in changes of proteins interactions. The modification of the substrate-linked ubiquitin with further ubiquitin molecules leads to the generation of polymeric ubiquitin chains. These chains can be linked through the N-terminus or through each of the seven lysine residues of ubiquitin, resulting in chains of different structure and function. K48-linked chains, for example, trigger degradation by the 26S proteasome, whereas K63-linked chains recruit binding partners into multimeric protein assemblies. For many other chain topologies, however, very little is known about substrates, enzymes, and functions.

 

We have discovered the K11-linked ubiquitin chain as an essential regulator of cell division in higher eukaryotes(Cell 2008). K11-linked ubiquitin chains are assembled during mitosis by the ubiquitin ligase APC/C, a finding we recently confirmed in cells using linkage-specific antibodies (Molecular Cell 2010a). We isolated the responsible E2 enzymes for K11-linkage formation, Ube2C/UbcH10 for chain initiation and Ube2S for chain elongation (PNAS 2009). We are currently investigating the mechanisms of specific and regulated K11-linked chain formation by these enzymes.

 

2. Ubiquitin-dependent regulation of proliferation and differentiation

 

Substrate specificity of ubiquitination depends on ~600-1000 so-called E3 enzymes, and for the majority of these enzymes, functions or substrates remain unknown. Ubiquitination often is reversible; the modification then has to be removed by one out of ~100 deubiquitinating enzymes (DUBs), most of which remain uncharacterized. We have developed a ubiquitin-related siRNA library and a robust siRNA-screening platform to isolate new E3s and DUBs with important roles in proliferation and differentiation. Using this setup, we could identify an important role for ubiquitin in regulating the composition and function of the spliceosome (Genes and Development, 2010). Loss of this pathway led to incorrect tubulin splicing, inaccurate spindle formation and reduced sensitivity to treatment with the chemotherapeutic taxol.

 

The identification of substrates of cell-cycle regulated ubiquitination is difficult. We have developed an in vitro-expression cloning/ubiquitination strategy to isolate new substrates of the E3 APC/C (Molecular Cell 2010b). These new substrates were spindle assembly factors required for Ran-dependent spindle formation. Their degradation was regulated by binding to importin-beta, which also inhibits their function in spindle formation. We are currently investigating the temporal regulation of the APC/C- and Ran-dependent degradation of spindle assembly factors.

 

3. Small molecule discovery to modulate the activity of ubiquitination enzymes

 

We are combining our biochemical insight and the siRNA-based enzyme discovery to identify targets for the development of small molecules against ubiquitination enzymes. We are screening for small molecules that could either active or inhibit the activity of these enzymes, providing a proof-of-principle that ubiquitination enzymes are attractive drug targets.

Selected Publications

Research papers:

 

Matsumoto M*, Wickliffe KE*, et al. (2010). K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody. Mol. Cell in press

 

Song EJ, Werner SL, Neubauer J, Stegmeier F, Aspden J, Rio D, Harper JW, Elledge SJ, Kirschner MW, and Rape M. (2010). The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome. Genes Dev. 24, 1434-47.

 

Song L, and Rape M. (2010). Regulated degradation of spindle assembly factors by the anaphase-promoting complex. Mol. Cell 38, 369-82.

 

Williamson A*, Wickliffe KE*, Mellone BG, Song L, Karpen G, and Rape M. (2009). Identification of a physiological E2 module for human APC/C. Proc. Natl. Acad. Sci. USA. 106(43):18213-8.

* These authors contributed equally to this work.

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.

 

Reviews:

Song, L., and Rape M. (2008). Reverse the curse: cell cycle regulation by deubiquitination. Curr. Opin. Cell. Biol. 20, 156-63.

 

Ye Y and Rape M. (2009). Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 10(11):755-64.

 

Rape M. (2009). Ubiquitin, infinitely seductive: symposium on the many faces of ubiquitin. EMBO Rep. 10(6):558-62.

 

Rape M. (2009). A set of surgical chain saws. EMBO J. 28(6):615-6.

 

Wickliffe K, Williamson A, Jin L, and Rape M. (2009). The Multiple Layers of Ubiquitin-Dependent Cell Cycle Control. Chem. Rev. Jan 15. 109(4):1537-48.

 

Williamson, A., Jin, L., and Rape, M. (2008). Preparation of synchronized human cell extracts to study ubiquitination and degradation. Methods Mol. Biol. 545:301-12.

 

* These authors contributed equally to this work.

 

Last Updated 2010-07-20