Open AccessTransactivation properties of c‐Myb are critically dependent on two SUMO‐1 acceptor sites that are conjugated in a PIASy enhanced manner
Author(s) -
Dahle Øyvind,
Andersen Tor Ø.,
Nordgård Oddmund,
Matre Vilborg,
Del Sal Giannino,
Gabrielsen Odd S.
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03504.x
Subject(s) - sumo protein , transactivation , myb , transfection , microbiology and biotechnology , gene knockdown , biology , immunoprecipitation , chemistry , transcription factor , biochemistry , gene , ubiquitin
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.