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

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 twohybrid 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.Keywords: c-Myb; transcription; SUMO-1; Ubc9; PIASy.The c-Myb transcription factor plays a central role in the regulation of cell growth and differentiation, in particular in hematopoietic progenitor cells (reviewed in [1]). Homozygous null c-Myb/Rag1 chimerical mice are blocked in early T-cell development, while mice with a c-myb null mutation display severe hematopoietic defects leading to in utero death at E15 [2,3]. The c-Myb protein consists of an N-terminal DNA-binding domain (DBD), a central transactivation domain (TAD) and a C-terminal negative regulatory domain (NRD). The DBD of c-Myb is comprised of the three imperfect repeats: R 1 , R 2 and R 3 , each related to the helix-turn-helix motif [4][5][6][7].Oncogenic alterations, as found in AMV v-Myb, include both N-and C-terminal deletions as well as point mutations [8]. AMV v-myb is a potent and cell-type specific oncogene that transforms target cells in the macrophage lineage and induces monocytic leukemia [8,9]. Several studies have attempted to define oncogenic determinants of v-myb. N-and C-terminal deletions remove several sites of protein modification, including an N-terminal CK2 phosphorylation site (S11 and S12) [10], and a putative MAPK-site (S528) [11][12][13] as well as acetylation sites [14,15] located in the deleted portion of the C-terminal NRD. In addition, specific point mutations in v-My...

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

“…However, the A 267 NAA M2 mutant had a dramatic effect and activated c-Myb more than 13-fold relative to the wild type. The activity of this mutant closely resembled that of the SUMO conjugation-negative 2KR mutant (Dahle et al, 2003), used as a positive control (Figure 2a). The v-Myb mimicking mutation I 267 NII had no such derepression effect; rather, it slightly lowered the activity of c-Myb.…”

“…Deletions in the v-myb gene eliminate the CRD (Figure 1a), making v-Myb behave like an activated form of c-Myb in many assays with lost SUMO conjugation playing a key role (Dahle et al, 2003). We recently realized that loss of SUMO conjugation sites might not be the only oncogenic alteration in v-Myb that relates to SUMO biology.…”

“…This leads to a severe drop in the activity of c-Myb (Bies et al, 2002;Dahle et al, 2003;Sramko et al, 2006). We have recently shown that this drop in activity is mainly caused by silencing of a SUMOregulated activation function in CRD, severely reducing the synergistic potential of c-Myb (Molvaersmyr et al, 2010).…”

Inhibition of Myb-dependent gene expression by the sesquiterpene lactone mexicanin-I

“…Expression vectors for v-Myb (pCDE26v-myb) and human c-Myb (pCIneo-HcM-HA) have been described. 23,26 v-Myb C130A is a v-Myb mutant with a substitution of cysteine 130 by alanine. The expression of endogenous mim-1 mRNA in HD11 cells transiently transfected with wild-type or C130A mutant v-Myb expression vectors was analyzed by northern blotting as described before.…”

FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci

“…To identify novel proteins that interact with the human c-Myb protein, we performed a yeast two-hybrid screening with full-length c-Myb as bait (Dahle et al, 2003). Among the positive interacting clones, we identified a sequence encoding the C-terminal part of human FLASH (or CASP8AP2; NM_012115).…”