Tarek A. Abbas, PhD
Genomic instability is the hallmark of cancer and thus, it is critical that we have a comprehensive understanding of the various players that promote and those that safeguard against genomic instability. Ubiquitin-dependent proteolysis plays a significant role in various physiological processes including cell cycle control and cellular proliferation and is frequently the target of oncogenic transformation. The specificity of protein substrate ubiquitylation is dictated by the activity of one of many E3 ubiquitin ligases either directly or through substrate recognition through adaptor proteins. Among these, the Cullin 4-based E3 ubiquitin ligase (CRL4) is emerging as a master regulator of cellular proliferation and genomic stability and is involved in multiple DNA repair processes. Cdt2/DTL, a WD-repeat containing protein associates with CRL4 (CRL4-Cdt2) and functions as a substrate recognition factor for recruiting substrates to the rest of the CRL4 ubiquitin ligase.
I have recently shown that CRL4-Cdt2 promotes the ubiquitin-dependent destruction of the cyclin dependent kinase (CDK) inhibitor p21, both in S-phase of the cell cycle and in response to UV irradiation (abbas et al., 2008). This activity is important for S-phase progression by virtue of its ability to free PCNA from inhibitory p21, as well as for optimal DNA repair by virtue of promoting PCNA-dependent translesion DNA synthesis (TLS). More recently, I found that the CRL4-Cdt2 ubiquitin ligase complex promotes the S-phase destruction of the histone H4 lysine 20 (H4K20) mono-methyl transferase Set8/Pr-Set7 and that this activity is important for optimal chromatin compaction and G2/M cell cycle progression (submitted to Cell).
Significantly, Cdt2 is frequently over-expressed in a variety of human tumors and its expression correlates with tumor grade, metastasis and poor survival. My current research aims at understanding how CRL4-Cdt2 impacts on genomic stability and contributes to cancer development. Specifically, I will A) Identify and characterize novel substrates for the CRL4-Cdt2 E3 ubiquitin ligase complex, and B) Test whether Cdt2 exhibits oncogenic activity in vivo. Using tap-tandem purification and mass-spectrometry analysis of Cdt2-associated proteins from human cells with or without DNA damage, I will identify new CRL4-Cdt2 substrates that may be involved in cellular proliferation and/or DNA repair. Standard biochemical techniques, including in vivo and in vitro ubiquitylation assays using purified CRL4-Cdt2 E3 ubiquitin ligase complexes, will verify whether the identified proteins are bona fide substrates. I will also generate transgenic mice over-expressing Cdt2 from Cre/LoxP constructs and test whether Cdt2 over-expression contributes to tumor development in animal model system. The results will advance our understanding of how to exploit the regulated process of protein ubiquitylation and proteolysis for cancer intervention purposes.