Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner.
The transcription factor v-Myb is a potent inducer of myeloid leukemias, and its cellular homologue c-Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999-9009) presented evidence that murine c-Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG-tagged SUMO-1. Here we provide independent evidence that human c-Myb is also subject to SUMO-1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV-1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two-hybrid screening revealed that the SUMO-1 conjugase Ubc9 is one of a few major Myb-interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO-1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb-target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb-induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c-Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c-Myb depends on the sumoylation sites located here.
Dahle
Øyvind
Ø
Department of Biochemistry, University of Oslo, Norway.
Andersen
Tor Ø
TØ
Nordgård
Oddmund
O
Matre
Vilborg
V
Del Sal
Giannino
G
Gabrielsen
Odd S
OS
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT; D017434:Protein Structure, Tertiary; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C485891:PIAS4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9