Abstract PTEN tumor suppressor plays crucial function in multiple cellular processes, including proliferation, migration, DNA repair, cell cycle and survival signaling. Its function is tightly controlled by a series of regulatory processes at transcriptional and post-translational levels. Small ubiquitin-related modifier (SUMO) is an essential post-transcriptional modifier which involves in many important biological functions. Previous studies showed that PTEN can be modified by SUMOylation on K254 and K266. SUMOylation on K254 controls the nuclear localization and retention of PTEN and plays essential role in DNA damage repair. On the contrary, SUMOylation on K266 is responsible for its membrane binding, which is required for the down-regulation of PI3K/AKT pathway. However, most studies mainly focus on SUMO1 modification, function of SUMO2/3 involved PTEN regulation is still unclear. In addition, the dynamic regulation and biological activities of different isoforms of SUMOylated PTEN in different subcellular compartments are not well-defined. Also, how PTEN phosphorylation impacted on PTEN SUMOylated have not been addressed fully. To address these questions, a series of PTEN SUMOylation defective-mutants targeted to different subcellular compartments were generated. In addition, a panel of phosphorylation sites mutants (T366A, S370A, S380A, T382A, T383A, and S385A) were included. These mutants were cotransfected with His-tagged SUMO1, SUMO2, and SUMO3 in 293T cells. SUMOylated PTEN protein species are affinity absorbed onto nickel columns. The characterization of these mutants will be discussed. To further delineate the dynamic regulation of SUMOylated PTEN, we are developing a BRET-based live cell imaging system by the ectopic co-expression of a HALO-tagged SUMO and NanoLuc-tagged PTEN expression constructs to monitor the subcellular localization of SUMO-conjugated PTEN in vivo. This work was supported by Hong Kong Research Grant Council (460713) and Hong Kong PhD Fellowship Scheme (PF-12-13876). Citation Format: Yubing Wang, Chiwai Wong, Mingfei Yan, Penelope Or, Andrew M. Chan. Mapping SUMOylation sites of PTEN tumor suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2015-2163