Mechanism and Functional Study of Sumoylation in Mammalian System

Our previous studies have demonstrated that human topoisomerase I (hTOP1) can form poly-SUMO1 conjugates. We also found that yeast Ubc9 can be sumoylated and after sumoylation, the conjugation activity of yeast Ubc9 as a result to be suppressed. In addition, we also observed that the sumoylation activity could be regulated by phosphorylation. The goals of this proposal are thus focused on the mechanistical study of the sumoylation reactions in vitro and the possible biological functions of the sumoylation of mammalian Ubc9 and using SUMO1 as a model molecule to study the cross-talk between phosphorylation and sumoylation. Three specific aims are proposed: A. To study the biological function of mammalian Ubc9 sumoylation. We observed that yeast Ubc9 can be sumoylated and the sumoylation of yeast Ubc9 leads yeast Ubc9 suppress its conjugating activity. However, Knipscheer et. al. reported that mammalian Ubc9 after sumoylation has different effects to different target proteins. Some proteins’ sumoylation will be increased, while another will be decreased. Our preliminary study showed that the sumoylation patterns of yeast Ubc9 and mammalian Ubc9 are different. This observation has prompted us to examine whether the difference in sumoylation pattern between yeast and mammalian Ubc9 results in different effects on their sumoylation activities This proposal is taken an effort to examine the role of mammamlian Ubc9 sumoylation. Three subjects of research are proposed: 1) To identify the sumoylation site(s) of mammamlian Ubc9. 2) To analyze whether the effect of sumoylation on mammamlian Ubc9 is depended on the site of sumoylation on Ubc9 to affect Ubc9 conjugating activity. 3) Using mammalian system to study the function of Ubc9 sumoylation. B. Using SUMO 1 as a model molecule to study the cross-talk between sumoylation and phosphorylation. We previous reported that sumoylation activity can be regulated by phosphorylation and we also observed that phosphorylation of SUMO1 by protein kinase A can enhance the sumoylation activity. We thus proposed to use SUMO 1 as a model molecule to study the cross-talk between sumoylation and phosphorylation. Three subjects of research are proposed. 1) To identify the phosphorylation site(s) of SUMO1 by protein kinase A. 2) To validate that the mutation on SUMO1 phosphorylation site(s) regulates the sumoylation activity of SUMO1. 3) To examine all other commercial available protein kinases whether they can phosphorylate SUMO1 and as a result to regulate the sumoylation activity of SUMO 1. C. To determine the major branch lysine residue(s) of SUMO1 for polysumoylation in vitro and examine the biological function of the SUMO1 branch linkages in vivo. Since different ubiquitin linkages associate with distinct cellular functions, this observation prompts us to examine whether SUMO linkages to its acceptor proteins reflect to their distinct cellular functions. To examine this possibility, three subjects of research are proposed. 1) To determine the major branch lysine residue(s) of SUMO1 for polysumoylation. 2) To construct shRNA-resistant SUMO1 cDNA and apply the shRNA-resistant cDNA to construct plasmid expressing SUMO1 with single site branch point. 3) To examine whether different SUMO1 linkage to its acceptor protein reflect to their distinct cellular functions.