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Ubiquitin-dependent Control of Cell Division and Differentiation

Research Summary

Michael Rape is interested in understanding principles of ubiquitin-dependent signaling and the roles of this posttranslational modification during cell division and differentiation. His work bridges the identification of critical ubiquitylation enzymes in cancer cells and embryonic stem cells with a biochemical dissection of the mechanisms that underlie the modification of important substrates. His studies provide insight into reactions that are required for normal development and whose misregulation causes several diseases, most notably cancer.

Posttranslational modification of proteins with the highly conserved ubiquitin, a reaction referred to as ubiquitylation, is essential for cell division, differentiation, and survival in all eukaryotes (Figure 1). During ubiquitylation, the carboxy-terminus of ubiquitin is attached to a substrate lysine, resulting in the formation of a covalent isopeptide bond. Transfer of a single ubiquitin often results in changes in substrate interactions, localization, or activity. The substrate-attached ubiquitin is, however, frequently modified with additional molecules to generate polymeric ubiquitin chains. Dependent on the attachment site used for chain formation, these chains adopt different structures and result in distinct consequences for the modified protein: K11- and K48-linked ubiquitin chains trigger substrate degradation by the proteasome, whereas K63-linked chains act as molecular glue, holding together large complexes that mediate DNA repair or NF-kB transcription factor activation. In my laboratory, we are interrogating the assembly and function of specific ubiquitin marks, dissecting their roles in proliferation and differentiation, and devising methods to alter ubiquitin-dependent signaling by small molecules. In short, we are attempting to decipher the ubiquitin code and reveal its roles in proliferation and differentiation.

Interrogating the Code: Assembly and Function of Specific Ubiquitin Modifications
Ubiquitylation requires a cascade of at least three enzymes, referred to as E1, E2, and E3. The human genome encodes two E1 enzymes with specificity for ubiquitin, ~40 E2s, and more than 600 E3s. E3s recruit specific substrates and activated ubiquitin, thereby promoting the modification of substrates with a particular ubiquitin mark. The biological outcome of ubiquitylation, therefore, depends on the substrate and linkage specificity of an E3-dependent ubiquitylation complex.

By analyzing products of an essential E3, the anaphase-promoting complex (APC/C), we discovered the function of an atypical chain linked through Lys11 of ubiquitin. We could show that K11-linked chains are specifically formed during cell division, consistent with their role in allowing cells to rapidly progress through mitosis. Using these novel chains as our model, we have uncovered key principles of linkage-specific chain formation, the reaction that allows cells to implement the ubiquitin code. We could show that rapid chain formation relies on substrate motifs, referred to as initiation motifs, which promote transfer of the first ubiquitin to a substrate lysine. Chain initiation is the rate-limiting step in K11-linked chain formation, and the responsible E2 Ube2C (also known as UbcH10) is tightly regulated. The short chains appended to a substrate by the APC/C and Ube2C are then rapidly extended by Ube2S, an APC/C-specific E2 that we discovered. By using an integrated approach combining structural, biochemical, and bioinformatic techniques, we could show that Ube2S achieves its linkage specificity by substrate-assisted catalysis, during which critical residues of the catalytic center are contributed by Ube2S and the ubiquitin molecule that contains the target lysine residue.

Our current work focuses on those aspects of the ubiquitin code that remain poorly understood, concentrating on three main questions:

  1. What are the mechanisms of ubiquitin chain elongation? We are particularly interested whether enzymatic requirements for assembling long chains are different from those leading to formation of the first linkage.
  2. What are functions of K11-linked chains? To answer this question, we have developed a novel technique, E3 reprogramming, that allows us to change the linkage specificity of a ubiquitylation enzyme in cells.
  3. What are functions of other uncharacterized linkages? To this end, we are searching for E3s with novel linkage specificity.
Determining the Functions of Ubiquitylation in Cell Division and Differentiation
Because only a few of the ~600 human E3s have been matched with substrates, the physiology of ubiquitin-dependent signaling remains poorly understood. However, it is well established that aberrant ubiquitylation can lead to tumorigenesis, indicating that this modification plays a central role in ensuring accurate cell division and differentiation. Therefore, we are identifying novel E3s that control cell division or differentiation. We are also interested in isolating other components or regulators of these enzymes as well as their crucial substrates. To achieve these goals, we use a plethora of techniques, such as high-content small interfering RNA (siRNA) screening, gene expression analysis, live cell imaging, and mass spectrometry, that are performed in somatic cells or human and mouse embryonic stem cells.

Our work has allowed us to reveal crucial mechanisms that establish proper cell division or morphology: For example, we showed that ubiquitylation drives the disassembly of mitotic checkpoint complexes, an important step before anaphase; we identified a deubiquitylating enzyme, the tumor suppressor Usp4, as a component that is required for activation of this mitotic checkpoint by chemotherapeutics; and we found proto-oncogenes whose levels are controlled by an intricate interplay between the APC/C and importin-β, a regulator of spindle formation. In addition, in stem cells, we uncovered a role for the essential E3 Cul3 and its adaptor, Klhl12, in controlling the size of COPII coats, a reaction that is important for the secretion of large proteins, such as collagen (Figure 2).

Our current studies are focused on the identification of E3s that play important roles in maintaining the pluripotency of human embryonic stem cells, a question we address by developing novel siRNA-screening platforms. We use similar approaches to isolate E3s that are important for cancer cell division, in the hope of pointing to targets for chemotherapy. In general, our unbiased approaches have served us well in providing insight into reactions that play fundamental roles in ensuring robust development and that are often misregulated in disease.

Altering the Code: Developing Chemical Tools to Inhibit or Activate Disease-Related Ubiquitylation Enzymes
We also plan to use our newly developed understanding of the mechanisms of ubiquitylation to identify small molecules that inhibit or activate disease-related ubiquitylation enzymes. Because many ubiquitylation enzymes do not possess a classical ATP-binding site, their targeting by small molecules likely requires new approaches. Using cullin-RING ligases and the APC/C as our model systems, we are building assay platforms, which are used in collaboration with the small-molecule discovery center at the University of California, San Francisco, to isolate active compounds.

Grants from the National Institutes of Health, the California Institute of Regenerative Medicine, the Shurl and Kay Curci Foundation, the Korean Institute of Science and Technology, and the Bakar Fellowship Program at the University of California, Berkeley, provided partial support for some of these projects.

As of September 3, 2013

Scientist Profile

University of California, Berkeley
Biochemistry, Cell Biology