PML nuclear bodies (NBs) consist of more than 80 constituents and are involved in many cellular processes such as apoptosis, DNA repair, cell cycle, transcriptional regulation and development. PML NBs are associated with the ‘nuclear matrix’ and vary in size from 0.3 to 1.0 !m in diameter. PML NBs came to prominence when PML, expressed as an oncogenic fusion protein with the retinoic acid receptor, was found in patients with acute promyelocytic leukaemia. PML is the major PML NB constituent and is involved in cellular activities such as apoptosis, growth suppression, antiviral defence and DNA repair, among many others. PML can be modified by the Small Ubiquitin-like MOdifier (SUMO) and contains a SUMO- interacting motif (SIM). Both SUMO-modification and SUMO-interaction play an important role in the formation of PML NBs.
The aim of this study is to gain deeper knowledge about PML, PML NBs and associated factors. Preliminary data show that PML is interacting with TRAF4- associated factor 1 (TFAF-1). The TRAF family of proteins forms part of a signalling pathway via the TNF receptor and is implicated in growth regulation. When TFAF-1 is expressed as a fusion protein with a red fluorescent protein it forms filamentous structures. These structures colocalise with death-effector filaments (DEFs). DEFs are formed by the Fas-associated death domain (FADD) and the prodomain of caspase-8. The main focus of this study was to discover more about how PML and PML NBs regulate apoptotic pathways, and thereby gain a better insight into the organisation of PML and PML NBs and how their disruption contributes to human disease. The association between TFAF-1, PML, PML NBs and DEFs was investigated further. DEFs were found to disrupt PML NBs and phosphorylation of FADD was found to be necessary for the disruption. Casein kinase 1 alpha (CK1 alpha) phosphorylates FADD at S194, which has been shown to regulate the apoptotic activities of FADD. Thus the regulation of the kinase activity and the phosphorylation of FADD, together with PML and PML NBs were investigated. Preliminary data shows that CK1 is modified by SUMO. This might take place at the PML NBs and adds a whole new aspect to the already very complex story of PML NBs and PML NB constituents.