Cope, Gregory Allan (2006) Regulation of SCF ubiquitin ligases by Jab1/Csn5 and the Cop9 signalosome. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-07222005-141530
SCF ubiquitin ligases regulate the ubiquitin-dependent proteolysis of a myriad of substrate proteins, including p27, Cyclin E, and IkBa. To further gain insight into SCF regulation and function, we purified SCF from mammalian cells and found the Cop9 Signalosome associated with SCF. Interestingly, deletion of the CSN in S. pombe resulted in the hypermodification of Cul1 with Nedd8 in vivo. Furthermore, we found that the CSN can promote the removal of Nedd8 from Cul1 in vitro, suggesting CSN regulates SCF through deneddylation. To investigate the basis of CSN-dependent deneddylation activity, we analyzed the CSN and the 26S proteasome for conserved sequences that could be representative of a catalytic motif. We identified the JAMM motif in Csn5 and Rpn11 of the proteasome. Mutations in JAMM eliminated CSN-dependent deneddylating activity. Moreover, mutations in JAMM reduce the restrictive temperature of several SCF temperature sensitive mutants, suggesting that CSN acts positively on SCF activity. Finally, a JAMM mutant failed to rescue Csn5D defects in drosophila. To investigate the biological effect due to loss of deneddylating activity, we constructed a stable cell line that expresses an inducible siRNA sequence toward CSN5. We found that knock-down of Csn5 results in a dramatic decrease in F-box protein levels. Moreover, this loss correlates with F-box protein substrate accumulation and hyperneddylation of Cul1. We propose an autocatalytic mechanism for the turnover of F-box proteins that is dependent upon the deneddylating activity of CSN.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Cop9; Csn5; Jab1; JAMM; proteasome; SCF; Signalosome; ubiquitin|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||20 July 2005|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||25 Jul 2005|
|Last Modified:||26 Dec 2012 02:55|
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