Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Xiang-Dong Zhang

Abstract

Roles of SUMOylation in cancer metastasis and mitotic progression has been elucidated in this thesis. This vital and dynamic post-translational modification has been known to be dysregulated in several kinds of cancer. Global SUMO-2/3 conjugation is upregulated in a mouse metastatic breast cancer cell line, 66cl4, compared to a non-metastatic cell line 168FARN from the same genetic background. In order to identify the SUMO-2/3 substrates, SILAC labeled lysates from the two cell lines were immunopurified using SUMO-2/3 monoclonal antibodies. By using SUMO-2/3 epitope peptide elution (SUMO-2/3-ePIPE) and quantitative mass spectrometry, 66 SUMO-2/3 targets were identified, of which 15 targets are upregulated/downregulated in 66cl4 compared to 168FARN and 45 substrates are linked to cancer. About ten of these substrates were validated using in vitro and in vivo SUMOylation assays. Further, overexpression of GFP-tagged SUMO-2/3 in 168FARN cells increased in 3D cell migration compared to control cells overexpressing GFP. These results clearly show a link between SUMO and cancer metastasis. As we know, uncontrolled cell division is a hallmark of cancer and therefore I focused on understanding the role of SUMO-2/3 modification in regulating cell cycle progression through mitosis in the second half of my thesis. Inhibition of global SUMOylation resulted in a prometaphase arrest and mislocalization of CENP-E from kinetochores. CENP-E has a SUMO interacting motif required for its kinetochore localization. We found that these mitotic defects in cells with inhibition of SUMOylation can be rescued by overexpressing Nuf2-SUMO2-SUMO2-SUMO2 fusion proteins, but not by Nuf2-SUMO2, Nuf2-SUMO2-SUMO2, and Nuf2-SUMO1-SUMO1-SUMO1 fusion proteins. Notably, we used these Nuf2 fusion proteins to mimic the various types of Nuf2 SUMOylation at kinetochores. We also demonstrated that Nuf2 is critical for CENP-E localization to kinetochores and also modified by polymeric SUMO chains both in vitro and in vivo. Importantly, we show that Nuf2-SUMO2-SUMO2-SUMO2 fusion protein has better binding affinity to CENP-E compared to Nuf2. These results suggest that trimeric SUMO-2/3 modification of Nuf2 represent the minimum and distinct signal for targeting CENP-E to kinetochores and mitotic progression in mammalian cells.

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