טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSimon Einav
SubjectMechanism of the Modulation of Substrate Recognition by a
CDK via Cyclin Phosphorylation
DepartmentDepartment of Medicine
Supervisors Professor Ami Aronheim
Professor Daniel Kornitzer
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The Cyclin-Dependent Kinases (CDKs) depend on the cyclin subunit for catalytic activity and, in some cases at least, for substrate selectivity. Cyclins are regulated at the level of synthesis, stability and subcellular localization. In this work we propose that by self-induced phosphorylation, cyclins can also be regulated at the level of substrate recognition.

The yeast (S. cerevisiae) CDK Pho85, in conjunction with the cyclin Pcl5, specifically phosphorylates the transcription factorGcn4, targeting it for degradation. The cyclin Pcl5 is itself also regulated via phosphorylation - induced degradation. In this work we identify the kinase that phosphorylates Pcl5 as Pho85. Kinetic analysis of the reaction indicates that Pcl5 is phosphorylated by the very Pho85 molecule that it activates.

In order to gain insight into the evolution of the Pcl5-Gcn4 interaction, we analyzed the homologous proteins in the pathogenic yeast Candida albicans. We found that like ScPcl5, CaPcl5 is rapidly degraded upon phosphorylation by Pho85, and identified both the sites of phosphorylation (Thr38 and Thr43) and the mechanism of degradation (SCFCaCDC4). By combining deletion analysis with phylogenetic comparison of Pcl5 sequences followed by site-directed mutagenesis, we localized the region of the cyclin involved in substrate recognition to the cyclin box domain, and specifically to the first helix in that domain.

Usinganalysis in vitro of the kinetics of the phosphorylation reaction of CaGcn4 by CaPcl5/Pho85, we further found thatphosphorylation of CaPcl5Thr38 and Thr43causes a loss of specific activity towards the cognate substrate CaGcn4,but has no effect on the catalytic activityof the kinase. We propose that phosphorylation induces an allosteric shift in the cyclin box structure that interferes with substrate recognition. This represents a novel mechanism of cyclin regulation, which imposes a time limit to the activity of a specific cyclin-CDK complex. In vivo, cyclin phosphorylation and inactivation is normally followed by degradation of the cyclin via SCFCaCDC4-mediated ubiquitination. This allowsre-association of Pho85 with a new cyclin molecule.