|Ph.D Student||Gildor Tsvia|
|Subject||Determinants of Substrate Recognition by a Cyclin-CDK|
Complex and an SCF Ubiquitin Ligase
|Department||Department of Medicine||Supervisor||Professor Daniel Kornitzer|
|Full Thesis text - in Hebrew|
The Cyclin-Dependent Kinases (CDKs) play key roles in various processes in the living cell. All cyclins have a common structural motif consisting of 5 a-helices, called the cyclin box (CB). The function of the cyclin subunit in activation of the kinase is well established, but it is likely that specific cyclins also participate in targeting the kinase to specific substrates. Pho85, a Saccharomyces cerevisiae homolog of the mammalian CDK5, is activated by up to ten cyclins (called Pcls: Pho85 Cyclins).
Gcn4 is a short-lived S. cerevisiae transcription factor. Its degradation depends on phosphorylation at residue Thr165 by Pho85 in conjunction with Pcl5, followed by ubiquitination by the ubiquitin-conjugating enzyme Cdc34 in conjunction with the ubiquitin ligase SCFCdc4. The pathogenic yeast Candida albicans contains a functional homolog of Gcn4. Here, we show that C. albicans Gcn4 (CaGcn4) is rapidly degraded and that this degradation depends on a Pho85 cyclin homolog, CaPcl5. The regulatory loop that includes Gcn4 and Pcl5 is conserved in C. albicans. However, the proteins have coevolved so that there is no cross-recognition between the cyclin homologs from the two species, providing the clearest evidence yet for a role for the cyclin in substrate selectivity. After phosphorylation on Thr222 by Pho85/CaPcl5 (the equivalent of Thr165 in ScGcn4), CaGcn4 is recognized in S. cerevisiae by the ubiquitin-conjugated-ligase SCFCdc4. Optimal recognition was established with the cognate species Cdc4, implying that the F-box protein and its substrate have also co-evolved. In C. albicans, CaGcn4 degradation depends on CaPcl5 and CaCdc34 but not on CaCdc4 or residue Thr222, suggesting that other phosphorylated residues (Thr86, Ser169 or Ser187) are recognized by one or more additional SCF complexes.
The cyclin CaPcl5 is phosphorylated in vivo and in vitro by Pho85. In vivo this phosphorylation causes rapid degradation of the cyclin. In vitro, we found that phosphorylated cyclin lost its specific affinity for CaGcn4, but not the ability to activate the CDK. A mutant of CaPcl5 in residue Val78, located in a predicted a-helix N-terminal to the CB (“helix 0”), similarly still activated the CDK, but lost its specific affinity for CaGcn4 both in vitro and in vivo. We found that the ScGcn4 recognition site in ScPcl5 is located in the CB, and assume that by analogy CaGcn4’s recognition site in CaPcl5 is located in the CB as well. Based on CDK5/p25 structure we propose that self-induced cyclin phosphorylation modulates specific substrate recognition, possibly via movement of Helix 0 against the CB.