Tài liệu Báo cáo khoa học: Transactivation properties of c-Myb are critically dependent on two

Transactivation properties of c-Myb are critically dependent

on two SUMO-1 acceptor sites that are conjugated

in a PIASy enhanced manner

Øyvind Dahle1

, Tor Ø. Andersen1

, Oddmund Nordga˚rd1

, Vilborg Matre1

, Giannino Del Sal2,3

and Odd S. Gabrielsen1

1

Department of Biochemistry, University of Oslo, Norway; 2

Laboratorio Nazionale CIB, Area Science Park, Trieste, Italy; 3

Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` degli Studi di Trieste, Italy

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 cotrans￾fected with FLAG-tagged SUMO-1. Here we provide

independent evidence that human c-Myb is also subject to

SUMO-1 conjugation under more physiological condi￾tions 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 fewmajor 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 sumoy￾lation 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]). Homozy￾gous null c-Myb/Rag1 chimerical mice are blocked in early

T-cell development, while mice with a c-mybnull 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 trans￾activation domain (TAD) and a C-terminal negative

regulatory domain (NRD). The DBD of c-Myb is com￾prised of the three imperfect repeats: R1, R2 and R3, each

related to the helix-turn-helix motif [4–7].

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 phosphoryla￾tion site (S11 and S12) [10], and a putative MAPK-site

(S528) [11–13] as well as acetylation sites [14,15] located in

the deleted portion of the C-terminal NRD. In addition,

specific point mutations in v-Myb abolish protein–protein

interactions [5], as well as phosphorylation as in the case of

V117D [16]. c-Myb has recently also been reported to be

subjected to SUMO-1 (small ubiquitin-related modifier)

conjugation [17].

The SUMO-1 protein is related to ubiquitin, but its

function, although presently unclear, seem to be other

than proteasomal degradation (reviewed in [18,19]). The

sequence homology between ubiquitin and SUMO-1 is

low, but the structures are highly similar [20], and they

use related conjugation mechanisms [21], including the

use of E3-like factors, which was recently identified for

sumoylation as the PIAS proteins (protein inhibitor of

activated STATs) [22–25]. The sequence YKXE has

been proposed as a consensus sequence for SUMO-1

conjugation [26]. The process of sumoylation is conserved

from yeast to man and is a dynamic and reversible

Correspondence to O. S. Gabrielsen, Department of Biochemistry,

University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway.

Fax: + 47 22 85 44 43, Tel.: + 47 22 85 73 46,

E-mail: [email protected]

Abbreviations: DBD, DNA-binding domain; MRE, Myb recognition

element; NRD, negative regulatory domain; PIAS, protein inhibitior

of activated STATs; SUMO-1, small ubiquitin-related modifier;

TAD, transactivation domain; Ubc9, ubiquitin conjugation enzyme 9.

(Received 5 December 2002, revised 31 January 2003,

accepted 6 February 2003)

Eur. J. Biochem. 270, 1338–1348 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03504.x