This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Cancer is one of the leading causes of death in the United States and in the world. Many cancers result from activation of proto-oncogenes or inactivation of tumor suppressor genes. In most cases, the precise mechanisms through which these oncoproteins/tumor suppressors work are poorly understood. Msp58 (microspherule protein 58 kDa), an oncoprotein, is evolutionarily conserved from fly to human and has been shown to play roles in various nuclear processes, including transcriptional regulation and mRNA export. Over-expression of Msp58 has been shown to induce cell transformation. The precise mechanism by which Msp58 carries out its normal nuclear functions is not known, nor is it understood why aberrant expression ofMsp58 leads to cellular transformation. This project aims to shed light on both of these questions.We have identified two novel interacting partners of Msp58: nucleoporin Tpr (translocated promoter region) and E3 ubiquitin ligase EDD (E3 identified by differential display). Tpr has been implicated in nuclear organization and in the nuclear export of protein and messenger RNAs (mRNAs). EDD has been shown toplay a critical role in cell proliferation and differentiation. We hypothesize that Msp58 carries out its functions by regulating the locations of some of its associating factors in the nucleus by, at least in part, interacting with Tpr; and EDD regulates the stability and/or the function of Msp58, directly or indirectly, through its ubiquitinligase activity. To test these hypotheses, in this project, we will characterize the interaction of Msp58 with Tpr and EDD, and determine the functions of these complexes. Deletion analyses followed by site-directed mutagenesis will be used to map the regions of the proteins that mediate their interactions. The locations ofproteins and mRNA will be determined by immunofluorescence confocal microscopy and immunoelectron microscopy. We will explore the functions of individual components of the complexes in cultured cells in which expression of the components will be altered, either silenced by RNA interference or overexpressedectopically. A long-term goal of this project is to explore how Msp58's over-expression leads to cellular transformation. Given that Msp58's interacting proteins, such as proliferation-associated nucleolar protein p120 and EDD, are often aberrantly expressed in various cancers, including hepatocellular carcinoma, andbreast, lung and ovarian cancers, this study, therefore, will not only advance our understanding of the fundamental cell biology underlying nuclear organization and nuclear transport, but also shed light on understanding the pathogenesis of cancers.
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