Study of Ubiquitinating Enzymes Essential for Cell Proliferation

Ubiquitination is a form of post-translational modification of proteins, in which the 76-residue ubiquitin polypeptide is conjugated to the lysine residues of either the substrate protein or the preceding ubiquitin molecule. Ubiquitination requires the sequential action of three types of ubiquitin enzymes: the ubiquitin activating enzyme (E1), the conjugating enzyme (E2) and the ligase (E3). In human genome, there are two different E1s, ~40 E2s and more than 600 E3s. Ubiquitination regulates a variety of cellular processes, including cell cycle progression, induction of inflammatory response, or intracellular protein trafficking. The anaphase-promoting complex/cyclosome (APC/C) and the SCF are two important E3 ligases regulating cell cycle progression. Understanding how they recognize their substrates and how their enzymatic activity is regulated will provide valuable clues for inhibiting unchecked cell proliferation such as in cancer. The objective of this dissertation is twofold: 1) Chapter 1: understanding how APC/C forms ubiquitin chains on its substrates. We found that APC/C preferentially uses lysine11 of ubiquitin to assemble ubiquitin chains on its substrates. By recognizing the same motif present in both the substrates and ubiquitin, APC/C switches from initial modification of the substrate to processive chain elongation, leading to the rapid turnover of its substrates. 2) Chapter 2 to 5: identifying Cul3Klhl12, a structural relative of SCF, as an essential ubiquitin ligase in embryonic stem cells (ESCs). In an siRNA screen, we found that Cul3Klhl12 affects cell adhesion and proliferation of ES cells by regulating COPII-mediated protein trafficking. Cul3Klhl12 ubiquitinates Sec31A, a coat protein of COPII vesicles, and causes the enlargement of the vesicles. This enables the COPII-dependent transport and proper deposition of collagen, a major component of extracellular matrix (ECM)