טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMeimoun Ariella
SubjectThe Roles of Kinases in the Degradation of the Yeast
Transcription Factor Gcn4
DepartmentDepartment of Medicine
Supervisor Professor Daniel Kornitzer


Abstract

The yeast transcriptional activator Gcn4 regulates a significant proportion of the yeast genes including amino acids and purines biosynthesis genes. One way to regulate the activity of Gcn4 is at the level of  protein degradation. It is known that Gcn4 is rapidly degraded through the ubiquitin pathway and is dependent on the ubiquitin-conjugating enzyme Cdc34 and on the ubiquitination complex SCFCdc4. Unlike many Cdc34/SCFCdc4 substrates which require phosphorylation by the cyclin dependent kinase Cdc28 prior to their ubiquitination, the kinase involved in Gcn4 degradation  has not been defined. Here we show that the cyclin dependent kinase Pho85 together with its Pcl5 cyclin partner are involved in Gcn4 phosphorylation and degradation in vivo. We also identified  the critical target site of Pho85 on Gcn4  at residue Thr165. Under conditions of amino acids limitation or inhibition of protein synthesis, conditions that stabilize Gcn4, we found that Gcn4 was not phosphorylated at Thr165, indicating that the Pho85 activity is inhibited under these conditions. Furthermore, we identified two important distinct recognition sites on Gcn4.  1) The Pho85 recognition site including the pThr165-Pro-Val-Leu residues. 2) A novel  SCFCdc4 recognition site that includes at least  four residues: Phe162-Leu163-Pro164-pThr165. The Pro, Val and Leu residues of the Pho85 recognition site, which are situated carboxy-terminal to this site, may be part of the recognition site of the SCFCdc4 complex as well.