CDC34 regulation and its role in vertebrate DNA replication initiation

Cell cycle progression and homeostasis require ubiquitin-dependent proteolytic degradation. Regulated protein degradation is a cascade of events involving the sequential activities of the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligating enzyme (E3). A requirement for the ubiquitin conjugating enzyme, CDC34, at the G1/S transition in eukaryotes has been well established. Suggesting a degradation requirement of critical substrates for the onset of DNA replication. The most characterized ubiquitin ligating enzyme that functions with CDC34 is p19S&barbelow;kp1/C&barbelow;ul1/p45Skp2 (F&barbelow;-box)/Roc1 (r&barbelow;ing) or simply the SCF/ring complex. The critical G1 to S phase regulators that must be degraded prior to the onset of S phase in vertebrates is currently unknown. Previous studies have shown that CDC34 in a large molecular size complex is required for the initiation of DNA replication in eggs from Xenopus laevis. We have partially purified and biochemically characterized the hCDC34 requirement for the onset of DNA replication in mammalian cells. Our studies suggest that the human CDC34 complex does not consist merely of CDC34 and SCF components, but may instead consist of previously unidentified CDC34-interacting proteins. We have taken several approaches to identify the putative novel associated proteins of CDC34 that are required for DNA replication activity. We have identified a novel CDC34 interacting protein, the regulatory subunit of Casein Kinase 2 (CK2) through a yeast two hybrid screen. We show that CDC34 is phosphorylated in vivo by a CK2 like kinase. We have mapped the phosphorylation sites to the last 36 amino acids of CDC34 in vitro and in vivo. We show that the last 36 amino acids hCDC34 are required for complementing the temperature sensitive phenotype in S. cerevesiae as well as supporting in vitro ubiquitination of a known mammalian substrate of CDC34, p27Kip1. Mutation of CK2-targeted residues within CDC34, S203, S222, S231, T233, and S236, abolished phosphorylation of CDC34 observed in vivo and markedly shifted nuclearly localized CDC34 into the cytoplasm. We have found that the phosphomutant of CDC34 is deficient in binding Cul1/Roc1 of the E3 complex. These results suggest a potential role for CK2-mediated phosphorylation in the regulation of CDC34 cell localization and function.