Target proteins of C/EBPαp30 in AML: C/EBPαp30 enhances sumoylation of C/EBPαp42 via up-regulation of Ubc9

Abstract

CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is a critical regulator for early myeloid differentiation. Mutations in C/EBP␣ occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominantnegative isoform (C/EBP␣p30). In the present study, using a global proteomics approach to identify the target proteins of C/EBP␣p30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBP␣p30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBP␣p30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBP␣p42 decreases the transactivation capacity on a minimal C/EBP␣ promoter. Importantly, overexpression of C/EBP␣p30 in granulocyte colony-stimulating factor (G-CSF)-stimulated human CD34 ؉ cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBP␣p30 target through which C/EBP␣p30 enhances the sumoylation of C/EBP␣p42 to inhibit granulocytic differentiation. IntroductionThe transcription factor CCAAT/enhancer-binding protein ␣ (C/ EBP␣) is crucial for granulocytic differentiation. [1][2][3] Alterations of the function of C/EBP␣ are a common feature of leukemic cells. 4,5 It was discovered in 10% of patients with acute myeloid leukemia (AML) that the CEBPA gene is mutated. 6,7 These mutations are found in AMLs with a myeloblast phenotype (French-AmericanBritish [FAB]-M1 and -M2 subtypes). The mutated gene results in the predominant expression of a 30-kDa protein initiated at an internal AUG start codon. This mutated isoform lacks the Nterminal transactivation domain 1 (TAD1). However, it possesses the intact bZIP protein-protein interaction domain and can interact with activators and repressors that affect its biological roles. The mutated 30-kDa isoform has been shown to act in a dominantnegative manner over the wild-type isoform. 5 The ratio of p30/p42 is critical for a physiologic granulopoiesis. 5,8 In contrast to C/EBP␣p42, C/EBP␣p30 fails to induce myeloid cell differentiation. It inhibits the expression of the endogenous granulocyte colony-stimulating factor (G-CSF) receptor and leads to an enhanced proliferation. 9,10 Recently, it was reported that C/EBP␣p30 directly interacts with the BCL2 promotor to fulfill this role. 11 Relatively little is understood about how C/EBP␣p30 exerts its dominant-negative effect over C/EBP␣p42 and how it inhibits C/EBP␣p42 during normal myeloid lineage development. We applied high-throughput proteomics to identify the target proteins of C/EBP␣p30. In our screen, we identified the ubiquitinconjugating enzyme (Ubc9) as a novel target of C/EBP␣p30.Ubc9 is an essential E2 enzyme required for small ubiquitinrelated modifier (SUMO) conjugation, or sumoylation. 12,13 Ubc9 is known to play a central role in su...

The presence of C/EBPα and its degradation are both required for TRIB2-mediated leukaemia

“…While our peptide mapping clearly shows specific sites in both TRIB2 and C/EBPα responsible for the direct interaction, in the absence of crystal structure information we cannot predict further based on our data the reason why p30 is not targeted by TRIB2 for degradation. Our data does not discount a role for p30 in TRIB2 mediated AML; p30 presence may, for example, activate a unique set of genes, inhibit other C/EBPs or bind to a unique set of proteins distinct from C/EBPα p42 as previously reported (32,33). Indeed, U937 cells ligases including β-TrCP, COP1 and Smurf1 (36).…”

Regulation of the sumoylation system in gene expression

“…Recently, it has been reported that the increased expression of a 30-kDa dominant-negative isoform (C/EBPαp30) of CCAAT/enhancer-binding protein α (C/EBPα), which is present in acute myeloid leukemia (AML) patients with mutations in Cebpα gene, can induce the transcriptional induction of Ubc9 [18]. But initial analysis failed to detect the direct association of C/EBPαp30 with a promoter region of Ubc9.…”

Translational Control in Myeloid Disease

“…High levels of expression of the p30 C/EBP protein have been shown to interfere with the DNA binding ability of the full length p42 C/EBP, thus inhibiting transactivation of key granulocytic target genes in a dominant-negative manner (Pabst et al, 2001), In addition, p30 transactivates the expression of a distinct subset of target genes different from that of the full length p42 C/EBP thereby altering their expression (Geletu et al, 2007). Mice engineered to express only the p30 C/EBP isoform resulted in the development of AML with complete penetrance (Kirstetter et al, 2008).…”