ORIGINAL ARTICLE The ubiquitin ligase Triad1 inhibits myelopoiesis through UbcH7 and Ubc13 interacting domains JA Marteijn 1,5,6 , LT van der Meer 1,6 , JJ Smit 2 , SM Noordermeer 1 , W Wissink 1 , P Jansen 1 , HG Swarts 3 , RG Hibbert 2 , T de Witte 1,4 , TK Sixma 2 , JH Jansen 1 and BA van der Reijden 1 1 Central Hematology Laboratory, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 2 Division of Biochemistry, The Netherlands Cancer Institute and Center for Biomedical Genetics, Amsterdam, The Netherlands; 3 Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and 4 Department of Hematology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Ubiquitination plays a major role in many aspects of hemato- poiesis. 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 poly- ubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respec- tively. 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. Leukemia (2009) 23, 1480–1489; doi:10.1038/leu.2009.57; published online 2 April 2009 Keywords: Triad1; myelopoiesis; ubiquitin; proteasome Introduction Ubiquitination plays a major role in many aspects of cell biology, including cell division, signaling, movement and apoptosis. 1 The conjugation of one or more ubiquitin peptides to a specific substrate protein can regulate the substrate’s activity or may result in its recognition and degradation by the 26S proteasome. In normal hematopoiesis, the ubiquitin proteasome route regulates the fate of many proteins, including, for example, growth factor receptor c-kit, cyclin D, Bcl-2 (B-cell CLL/lymphoma 2) and transcription factors, such as GATA-2, acute myeloid leukemia-1 and Gfi1 (growth factor independence 1). 2 Deregulation of the ubiquitin proteasome route is also implicated in malignant hematopoiesis. 2 For example, the von Hippel–Lindau (VHL) protein is responsible for poly-ubiquitination and subsequent degradation of the Hif1a (hypoxia-inducible factor 1, a subunit) protein. Mutations in VHL that are found in polycythemia patients result in defective ubiquitination that causes stabilization of Hif1a. As a result, expression of hypoxia-induced genes, including erythropoietin, increases. High erythropoietin levels are believed to contribute to the high red blood cell counts that are found in congenital polycythemia. 3 In Fanconi anemia, mono-ubiquitination of the Fanconi anemia D2 protein is inhibited, resulting in a defect in the DNA repair mechanism. This results in genetic instability that predisposes Fanconi anemia patients to develop malig- nancies such as acute myelogenous leukemia. 4 Overexpression of MDM2, as found in many leukemia patients, results in increased poly-ubiquitination and degradation of the tumor suppressor protein, p53. 5 Recently, acquired mutations in Cbl (Casitas B-cell lymphoma) proteins were identified in acute myelogenous leukemia. Cbl is involved in the ubiquitination and subsequent internalization and degradation of the activated Flt3 (fms-related tyrosine kinase 3) receptor. As a result of mutations in Cbl, the Flt3 receptor is no longer ubiquitinated and thereby no longer inactivated, resulting in a constitutive active receptor. 6,7 Although it is clear that ubiquitination plays a crucial role in regulating the activity of many important proteins that control hematopoiesis, little is known about the enzymes that facilitate ubiquitination. The covalent coupling of ubiquitin to a substrate protein requires subsequent action of three enzymes. First, the E1 enzyme activates ubiquitin, which is then transferred to an E2 ubiquitin-conjugating enzyme (Ubc). In concert with Ubcs, ubiquitin E3 ligases catalyze the final step by facilitating the covalent attachment of ubiquitin to a substrate protein or the attachment of a ubiquitin to the preceding ubiquitin to form poly-ubiquitin chains. The E3 ligases are responsible for the specificity of the ubiquitination process as they recognize the substrates. 8 We have described earlier Triad1 as a ubiquitin ligase that regulates myelopoiesis through its ubiquitin ligase activity. 9 Expression of Triad1 is induced during myeloid differentiation and forced expression results in the inhibition of proliferation of immature murine bone marrow cells. Triad1 is the founding member of a subclass of ubiquitin ligases that contain two RING (really interesting new gene) finger domains. The RING finger containing E3 ligases represent the largest family of the group of proteins that have ubiquitin ligase activity. This family can be subdivided in E3 ligases that function as Received 27 May 2008; revised 29 January 2009; accepted 23 February 2009; published online 2 April 2009 Correspondence: Dr BA van der Reijden, Central Hematology Laboratory, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: B.vanderreijden@chl.umcn.nl 5 Current address: Department of Genetics, ErasmusMC, Rotterdam, The Netherlands. 6 These authors contributed equally to this work. Leukemia (2009) 23, 1480–1489 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu