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Access Type

WSU Access

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Xiang-Dong Zhang

Second Advisor

Karen Beningo

Abstract

SUMOylation is an essential post-translational modification that regulates a variety of critical cellular pathways ranging from nuclear transport to protein stability. Accumulating lines of evidence have shown that a perturbation of the SUMOylation pathway is associated with human diseases, especially various types of cancer. Our recent proteomic studies revealed a drastic increase in levels of SUMO2/3 modification on the proinflammatory cytokine MIF in the metastatic breast cancer cell line compared to the non-metastatic control cell line. Interestingly, the increase in levels of both MIF and global SUMO-2/3 modification in the metastatic cells are positively correlated to that of unmodified MIF proteins when compared to the non-metastatic control cells. Furthermore, global inhibition of SUMOylation by overexpression of the SUMO-specific isopeptidase SENP2 greatly decreases levels of MIF proteins. In addition, we found that endogenous MIF has a half-life of about three hours and is degraded through the proteasomal pathway. Moreover, global inhibition of SUMOylation by SENP2 overexpression significantly reduce the stability of MIF proteins. Furthermore, the lysine 78 residue of MIF is required for its SUMOylation in vitro. Importantly, we showed that MIF has a specific interaction with the SUMO E3 ligase PIAS3, which has been previously known to be upregulated in various types of cancer. In addition to my studies of SUMOylation in regulating the stability of MIF, which is known to promote tumorigenesis and metastasis through upregulation of cell proliferation and invasion, my graduate studies had also focused on elucidating how SUMOylation of the Ran GTPase activating protein RanGAP1 is regulated. As a key regulator of nuclear transport, RanGAP1 along with other important players in this process are often upregulated in various types of cancer. We demonstrated that RanGAP1 shuttles between the nucleus and the cytoplasm in mammalian cells, and that the exportin CRM1 mediates its nuclear export. Additionally, the NPC-associated SUMO1-modified RanGAP1 is highly stably associated with the cytoplasmic filaments of the nuclear pore complex (NPC) and requires longer hours to redistribute into the nucleoplasm in cells with inhibition of the CRM1-mediated export when compared to the cytoplasmic unmodified RanGAP1. The C-terminal 541-589 amino acid region of RanGAP1 is crucial for its nuclear import and the 26 residue region (541-566) within this C-terminus contains a functional nuclear localization signal. We also demonstrated that SUMOylation of RanGAP1 is independent of SUMO E3 ligase RanBP2. Our studies have focused on SUMOylation of these two important proteins MIF and RanGAP1, both of which are intricately associated with tumorigenesis and metastasis. As the relative distribution of MIF between the nucleus and the cytoplasm is an important prognostic determinant in cancer progression, it would be interesting to investigate the role of RanGAP1 in the nucleocytoplasmic transport of MIF proteins. The further studies of these two proteins and their SUMOylation in the future may provide the important information that may lead to novel therapeutic treatment of human cancers.

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