SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation

PML is a nuclear phosphoprotein that was first identified as part of a tran slocated chromosomal fusion product associated with acute promyelocytic leu kaemia (APL). PML localises to distinct nuclear multi-protein complexes ter med ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), w hich are disrupted in APL and are the targets for immediate early viral pro teins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and I kappa B alpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degra dation. The precise molecular role for the SUMO-1 modification of PML is un clear, and the specific lysine residues within PML that are targeted for mo dification and the PML subdomains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger d omain. Using site-specific mutagenesis, we have identified the primary PML- SUMO-1 modification site as being part of the nuclear localisation signal ( Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuc lear localisation as only nuclear PML is modified. The sequence of the modi fication site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1, We show that SUMO-1 modification appears to be dependant on th e correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RAR A. Our results provide significant insights into the role of SUMO-1 modific ation of PML in both normal cells and the APL disease state.