SV40 large T antigen (T Ag) has identified many important cellular factors that play key roles in SV40 transformation as well as in normal cellular growth and cancer. CUL7 (p185) was identified as a T Ag associated protein and provides a critical role in T antigen transformation and cellular growth. CUL7 is a member of the Cullin RING ligase family that targets specific protein substrates for ubiquitination and subsequent degradation. CUL7 binds specifically to FBXW8 to form an SCF-like (SKP1-Cullin-F box) E3 ubiquitin ligase. This proposal seeks to determine the molecular basis for the CUL7 contribution to T Ag transformation, cellular growth and cancer. The central hypothesis is that CUL7 and FBXW8 serve as tumor suppressors that play an essential role in growth and T Ag transformation. This hypothesis is based on the following observations. Homozygous loss of CUL7 results in the human 3-M syndrome characterized by severe short-stature. Similarly, loss of Cul7 or Fbxw8 in mice results in severe growth defects that resemble the 3-M syndrome. Importantly, loss of Fbxw8 increases the incidence of intestinal polyps in the APCMin mouse model of cancer. In addition, SV40 T Ag transformation appears to inactivate CUL7 function. Loss of Cul7 or Fbxw8 appears to promote genomic instability and increase the frequency of aneuploidy and aberrant centrosome number. To determine the molecular basis for CUL7's contribution to T Ag transformation, growth and cancer, the following specific aims are proposed.
Specific Aim 1 : Determine how CUL7 E3 ubiquitin ligase activity is regulated by cellular factors and by SV40 large T Ag. Recombinant CUL7, SKP1, RBX1 and FBXW8 form an SCF-like complex with E3 activity. The impact of T Ag on CUL7 E3 activity will be assayed.
Specific Aim 2 : Identify CUL7-FBXW8 substrates. CUL7 has substrate-independent E3 ubiquitin ligase activity and FBXW8 serves to target specific substrates for ubiquitination. Substrates will be identified using proteomic and high throughput screens and validated using conditional knockout cells and T antigen transformation assays.
Specific Aim 3 : Determine role of CUL7 and FBXW8 in tumorigenesis and T Ag transformation. The consequences of Cul7 and Fbxw8 loss on tumor formation will be evaluated using conditional and constitutive knockout mouse strains including APCMin model of intestinal polyps and Trp53 mutant mice. In addition, we will determine if T Ag's ability to induce chromosomal instability is dependent, at least in part, on perturbation of CUL7 and FBXW8 function.
The DNA tumor virus SV40 has led to the discovery of many important tumor suppressor genes including the retinoblastoma gene and p53. This proposal seeks to determine if CUL7 and FBXW8 are tumor suppressor genes that are important for normal growth and SV40 induced cellular oncogenesis.
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