SUMO modification regulates the transcriptional activity of FLASH.
FLASH is a huge multifunctional nuclear protein that has been linked to apoptotic signalling, transcriptional control and Cajal body function. To gain further insight into the functions of the FLASH protein, we performed a yeast two-hybrid screening with FLASH as bait and identified the SUMO-conjugating enzyme Ubc9 as an interaction partner. The main interaction surface for Ubc9 was found in the C-terminal part of FLASH, which is also a target for sumoylation. We identified K1813 as the major sumoylation site in FLASH, being enhanced by the SUMO E3 ligases Pc2 and PIASy. Disruption of this SUMO-conjugation site did not change the speckled subnuclear localization of FLASH, but it caused a reduction in FLASH activity as measured in a Gal4-tethering assay. Interestingly, the SUMO-specific protease SENP1 activated FLASH in the same assay. Overall, our results point to a complex involvement of sumoylation in modulating the function of FLASH.
Alm-Kristiansen
Anne Hege
AH
Department of Molecular Biosciences, University of Oslo, Blindernveien 31, Blindern, Oslo, Norway.
Norman
Ingrid Louise
IL
Matre
Vilborg
V
Gabrielsen
Odd Stokke
OS
D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D051017:Apoptosis Regulatory Proteins; C485604:CASP8AP2 protein, human; D002135:Calcium-Binding Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine