Ubiquitin modifications are increasingly recognized as key regulatory events in basic cell biology processes that impact the development of cancer, including cell cycle and cell survival versus cell death decisions. This application focuses on the role of a new isoform of the SCF ubiquitin ligase F-box protein Skp2, termed Skp2B, which we recently identified, in the regulation of mammary tumorigenesis. Skp2 is a nuclear protein that acts as an adaptor between substrates for ubiquitination and the core of the SCFSkp2 ubiquitin ligase complex. Skp2 is overexpressed in a variety of cancer types and has been linked to cancer progression. We have isolated Skp2B, a splice variant that localizes to the cytoplasm. We found that Skp2B does not act as a dominant negative form of Skp2. Further, we found that Skp2B is overexpressed in a large number of primary breast cancers, and that its overexpression leads to mammary carcinoma in an MMTV-Skp2B transgenic model and acceleration of the growth of human breast cancer xenografts in nude mice. Further, immunohistochemical analysis of Skp2 using antibodies that recognize both Skp2 and Skp2B, revealed that in a large number of breast cancer samples, Skp2 staining is cytoplasmic suggesting that Skp2B rather than Skp2 is overexpressed in these cancers. In terms of its function, we found that Skp2B localizes to the mitochondria and we have isolated a subset of mitochondria proteins that interact specifically with Skp2B by a yeast two-hybrid screen. Of particular interest is the mitochondrial chaperone BAP37 that faces the inter membrane space (IMS), where several pro-apoptotic proteins reside. Importantly, we found that BAP37 associates with ubiquitinated proteins that Skp2B promotes the ubiquitination of a misfolded IMS protein and that inhibition of Skp2B by shRNA induces spontaneous apoptosis. These novel findings provide the foundation of our hypothesis that: Skp2B interaction with BAP37 underlies a quality control that monitors the presence of misfolded IMS proteins and targets their elimination via ubiquitination. Further, that Skp2B overexpression protects breast cancer cells from IMS stress-induced apoptosis. We will test our hypothesis using our MMTV-Skp2B transgenic mice model, by engineering a misfolded IMS protein and test its ability to be ubiqutinated by Skp2B, finally we will isolate the ubiquitin ligase in association with Skp2B.
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