The ubiquitin ligase Triad1 inhibits myelopoiesis through UbcH7 and Ubc13 interacting domains

Abstract

Ubiquitination plays a major role in many aspects of hematopoiesis. Alterations in ubiquitination have been implicated in hematological cancer. The ubiquitin ligase Triad1 controls the proliferation of myeloid cells. Here, we show that two RING (really interesting new gene) domains in Triad1 differentially bind ubiquitin-conjugating enzymes, UbcH7 and Ubc13. UbcH7 and Ubc13 are known to catalyze the formation of different polyubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respectively. In line with the dual Ubc interactions, we observed that Triad1 catalyzes the formation of both types of ubiquitin chains. The biological relevance of this finding was studied by testing Triad1 mutants in myeloid clonogenic assays. Full-length Triad1 and three mutants lacking conserved domains inhibited myeloid colony formation by over 50%. Strikingly, deletion of either RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. We conclude that Triad1 exhibits dual ubiquitin ligase activity and that both of its RING domains are crucial to inhibit myeloid cell proliferation. The differential interaction of the RINGs with Ubcs strongly suggests that the ubiquitination mediated through UbcH7 as well as Ubc13 plays a major role in myelopoiesis.

HoxA10 Influences Protein Ubiquitination by Activating Transcription of ARIH2, the Gene Encoding Triad1

“…The E3 ligase attaches ubiquitin to the substrate, thereby tagging the substrate for proteasomemediated degradation. Previous studies determined that Triad1 interacts functionally with several E2 ligases, including Ubch7 and Ubc13 (19,20). Co-overexpression of Triad1 and UbcH7 increases total protein ubiquitination in myeloid progenitor cells or myeloid cell lines, but genuine Triad1/UbcH7 substrate proteins have not been identified (20).…”

“…Previous studies determined that Triad1 interacts functionally with several E2 ligases, including Ubch7 and Ubc13 (19,20). Co-overexpression of Triad1 and UbcH7 increases total protein ubiquitination in myeloid progenitor cells or myeloid cell lines, but genuine Triad1/UbcH7 substrate proteins have not been identified (20). Although Triad1 is ubiquitously expressed, expression increases during myelopoiesis and is greatest in mature neutrophils (19).…”

Identification of Ubiquitin System Factors in Growth Hormone Receptor Transport

“…Triad1 is an E3 ligase that has 2 RING domains and, consequently, has the ability to bind two ubiquitin conjugases. Previously, Marteijn and co-workers identified both Ubc13 and UbcH7 as E2s involved in Triad1 action (Marteijn et al, 2009). As seen in Fig.…”

“…Cell cycle, cell growth (Marteijn et al, 2009) CHFR* CHFR Cell cycle (Bothos et al, 2003, Loring et al, 2008 PARK2* Parkin Neuroprotection, cell death (Lo Bianco et al, 2004) RNF8* RNF8 DNA repair (Plans et al, 2006) Rad5 Rad5 DNA repair (Torres-Ramos et al, 2002) Rad18 PCNA DNA repair (Ulrich & Jentsch, 2000) STUB1* CHIP Heat shock system TRAF2 Traf2 TNF signalling (Habelhah et al, 2004) TRAF6 Traf6 TNF signalling (Wooff et al, 2004) Furthermore, these K63-linked ubiquitin chains have been implied in endocytosis in yeast (Lauwers et al, 2009) and in mammalian cells (Kamsteeg et al, 2006). To investigate whether K63-linked ubiquitylation is indeed involved in GH receptor endocytosis, we selected nine E3 ubiquitin ligases that are known to interact with Ubc13 (listed in Table 3) (Bothos et al, 2003, Deng et al, 2000, Loring et al, 2008, Marteijn et al, 2009, Plans et al, 2006, Sun et al, 2004, Torres-Ramos et al, 2002.…”

“…To investigate whether K63-linked ubiquitylation is indeed involved in GH receptor endocytosis, we selected nine E3 ubiquitin ligases that are known to interact with Ubc13 (listed in Table 3) (Bothos et al, 2003, Deng et al, 2000, Loring et al, 2008, Marteijn et al, 2009, Plans et al, 2006, Sun et al, 2004, Torres-Ramos et al, 2002. As gene silencing of Traf2 and Traf6 induced cell death in previous experiments (data not shown), and rad5 and rad18 are mainly involved in DNA repair, we excluded them from further investigation and performed gene silencing experiments for the 5 remaining E3 ligases using validated siRNAs (Table 3, asterisk).…”