The ubiquitin-conjugating enzyme Cdc34 plays an essential role in promoting the G1-S phase transition of the eukaryotic cell cycle and is phosphorylated in vivo. In this study we investigated if phosphorylation regulates Cdc34 function. We mapped the in vivo phosphorylation sites on budding yeast (S207 and S216) and human Cdc34 (S203, S222 and S231) to serine residues in the acidic tail domain, a region which is critical for Cdc34's cell cycle function. Protein kinase CK2 phosphorylates both yeast and human Cdc34 on these sites in vitro. CK2-mediated phosphorylation increased yeast Cdc34 ubiquitination activity towards S. cerevisiae Sic1 in vitro, when assayed in the presence of its cognate SCF Cdc4 E3 ligase. Similarly, mutation of the Cdc34 phosphorylation sites to alanine, aspartate or glutamate altered Cdc34-SCF Cdc4-mediated Sic1 ubiquitination activity. Similar results were obtained when Cdc34's ubiquitination activity was assayed in the absence of SCF Cdc4, indicating that phosphorylation regulates the intrinsic catalytic activity of Cdc34. To evaluate the in vivo consequences of altered Cdc34 activity, wild-type Cdc34 and the phosphosite mutants were introduced into a S. cerevisiae cdc34 deletion strain, and following synchronization in G 1 phase, progression through the cell cycle was monitored. Consistent with the increased ubiquitination activity in vitro, cells expressing the phosphosite mutants with higher catalytic activity exhibited accelerated cell cycle progression and Sic1 degradation. These studies demonstrate that CK2-mediated phosphorylation of Cdc34 on the acidic tail domain stimulates Cdc34-SCF Cdc4 ubiquitination activity and cell cycle progression.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|