Maintenance of an intricate balance between positive and negative regulators of the cell cycle is vital, as altered cell cycle regulation results in the uncontrolled cell division that leads to cancer. Cul3 is a novel type of SCF-like ubiquitin ligase that targets proteins for degradation by the ubiquitin-mediated proteolysis system. Cul3 binds to BTB domain-containing proteins which serve as adaptor molecules, also binding the substrate for degradation. In our preliminary work, a yeast two-hybrid screen for proteins that interact with mammalian Cul3 identified nine BTB domain-containing proteins, known as Cullin three binding proteins, or Ctbs. More in depth analysis of one, Ctb9/KLHDC5, has begun to elucidate a role for Cul3 in the regulation of cytoskeletal structure. The work described here shows targeting of the microtubule severing protein, p60/katanin by the Cul3 complex using Ctb9/KLHDC5 as a specific substrate adaptor. Cells that are deficient in Cul3 accumulate excess p60/katanin, and are unable to complete mitosis normally, resulting in the accumulation of binucleated cells. This work represents a novel way in which Cul3 activity regulates the cell cycle by controlling microtubule dynamics during mitosis.

A major substrate of Cul3 is cyclin E. Cyclin E regulates the G1 to S transition through its interaction with Cdk2. Elevated levels of cyclin E protein have been detected in breast cancers, and such increased expression is correlated with a poorer prognosis. However, cell lines derived from human breast tumors rarely show increased cyclin E mRNA levels, implying the changes are occurring posttranscriptionally. Preliminary studies in our laboratory have shown that decreased expression of Cul3 is correlated with increased levels of cyclin E protein in human breast tumors, suggesting that altered Cul3 activity may be responsible for the accumulation of cyclin E. The work presented in this thesis describes the development of a model system to determine the role Cul3 plays in breast cancer. Targeted deletion of Cul3 from mouse mammary tissue results in hyperplasia of the mammary ducts and increased cyclin E protein levels in the tissue. These conditions are likely to lead to a malignant state.

Based on this work and the work of others, we propose that Cul3 acts as a tumor suppressor by targeting for degradation multiple proteins involved in cell cycle regulation. We suggest that analysis of Cul3 expression could potentially be developed as a marker for the early stages of transformation in breast tissue, before excessive tumor growth occurs. Not only is this work relevant to breast cancer, but could be important in other types of cancer as cyclin E as well as other Cul3 substrates are expressed in all tissues.