In the last two decades there has been an exciting convergence in studies of the cell cycle and of oncogenesis. It is now clear that deregulation of the cell cycle machinery contributes to the uncontrolled proliferation and genomic instability typical of tumor cells. The cell division cycle is controlled by the sequential activation of various cyclin-dependent kinases (CDKs). The ubiquitin-proteasome proteolysis pathway is a major mechanism by which extracellular mitogens and anti-mitogens control CDK activity. Proteolysis of many cell cycle regulators is controlled by SCF ubiquitin ligases, each formed by four subunits: Skp1, Cul1, Roc1 and one of many F-box proteins. The substrate specificity of SCFs is thus determined by distinct F-box proteins that act as substrate recognition factors.

One of the particular interests of our laboratory has been the study of the CDK-inhibitors p21 and p27, which we have shown to be substrates of the ubiquitin system. The degradation of nuclear p21 and p27 allows the activation of CDKs, which in turn is essential for DNA replication to occur. We have demonstrated that CDK-mediated phosphorylation of p27 on threonine 187 is essential for p27 ubiquitinylation, and that the F-box protein Skp2 is a rate-limiting component of the molecular machine that ubiquitinylates and degrades p21 and p27. Skp2 requires a physical association with Cks1 (CDK subunit 1) to recognize these two substrates. Skp2 and Cks1 proteins are unstable in G1, and their degradation prevents unscheduled degradation of p21 and p27, and premature entry into S-phase. Significantly, degradation of Skp2 and Cks1 during G1 is controlled by another cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C).

We have expanded the studies on p27 to the clinic and found that destabilization of p27 correlates with tumor aggressiveness and poor prognosis in human epithelial cancers and lymphomas. Significantly, Skp2 levels inversely correlate with p27 expression in human breast cancers and in human lymphomas, and Skp2 cooperates with activated N-Ras in an in vivo model of lymphomagenesis.

Using human Skp1 as a bait in a two-hybrid screen and searching DNA databases we have identified and characterized a family of 26 novel human F-box proteins. We investigated further the function of one of these F-box proteins, ß-Trcp1, by inactivating its gene in mice. This knockout exposes an unexpected and crucial role for ß-Trcp in regulating the timely order of meiotic and mitotic events. ß-Trcp1-/- males show reduced fertility correlating with an accumulation of metaphase I spermatocytes. ß-Trcp1-/- MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles and misaligned chromosomes. Furthermore, we demonstrated that Emi1, an inhibitor of APC/C, is a substrate of ß-Trcp1. In contrast, stabilization of interphase substrates such as ß-catenin, IKB and Cdc25A does not occur in the absence of ß-Trcp1 and instead requires the additional silencing of ß-Trcp2 by siRNA.

In summary, the long-term goal of our research is to study how F-box proteins control the mammalian cell division cycle. Our laboratory is using an interdisciplinary approach that includes cellular biology, mouse genetics and biochemical methods. As we continue to discover the details of how timed degradation of cellular regulatory proteins by the ubiquitin-system controls growth and proliferation, we have begun to integrate our basic research with an understanding of malignant transformation. We anticipate that the results of our studies will have a broad impact in both basic science and translational cancer biology.