Transactivation properties of c-Myb are critically dependent on two SUMO-1 acceptor sites that are conjugated in a PIASy enhanced manner

Eur J Biochem. 2003 Mar;270(6):1338-48. doi: 10.1046/j.1432-1033.2003.03504.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Carrier Proteins / metabolism*
  • Cell Line
  • Humans
  • Intracellular Signaling Peptides and Proteins*
  • Ligases / metabolism
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / metabolism
  • Poly-ADP-Ribose Binding Proteins
  • Protein Inhibitors of Activated STAT
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-myb / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • SUMO-1 Protein / metabolism*
  • Transcriptional Activation*
  • Two-Hybrid System Techniques
  • Ubiquitin-Conjugating Enzymes*

Substances

  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • Nuclear Proteins
  • PIAS4 protein, human
  • Poly-ADP-Ribose Binding Proteins
  • Protein Inhibitors of Activated STAT
  • Proto-Oncogene Proteins c-myb
  • Recombinant Fusion Proteins
  • SUMO-1 Protein
  • Ubiquitin-Conjugating Enzymes
  • Ligases
  • ubiquitin-conjugating enzyme UBC9