Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding

Abstract

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for IntroductionTribbles (Trib) pseudokinases have recently emerged as important contributors to dysregulated signaling in malignant hematopoiesis. All 3 mammalian Trib homologs, Trib1, Trib2, and Trib3 (collectively termed "Trib1-3"), are associated with human malignancies. [1][2][3] Trib1 and Trib2 function as oncogenes in acute myelogenous leukemia (AML) and cooperate with homeobox gene 9 (HoxA9) to accelerate the onset of AML in murine AML models. 2,4 In particular, Trib2 is highly expressed in a specific subset of human AMLs that are associated with impaired CCAAT/ enhancer-binding protein-alpha (C/EBP-␣) function. 2 These AMLs express both myeloid and T-cell markers, and some contain activating Notch1 mutations. 5 As Trib2 is a direct transcriptional target of oncogenic Notch1, we hypothesize that the oncogenic Notch1 mutations are activating Trib2 expression in some of these tumors. 5 These biphenotypic AMLs respond poorly to current therapies, suggesting that new therapeutic strategies are needed. 6 Trib proteins interact with various signaling molecules and transcription factors, including activating transcription factor 4 (ATF4), 7 p65, 8 C-terminal interacting protein (CtIP), 9 mitogenactivated protein kinase kinase (MAPKK), 10 protein kinase B (AKT), 11 and constitutive morphogenesis 1 (COP1). 12 We recently demonstrated that Trib2 binds to and facilitates the degradation of C/EBP-␣, and proposed that this process is a key feature of Trib2-induced AML. 2 Trib2 (also termed c5Fw, TRB-2, GS3955, TRB2, and AW319517) has 3 clearly distinguishable regions, including an N-terminal portion, a central serine/threonine kinase-like domain (KD), and a C-terminal region that contains a COP1-binding site. The Trib2 N-terminus is the most divergent region of mammalian Trib proteins and is characterized by a high serine and proline content. The C-terminus contains a DQxVPx motif that is shared by Trib1-3 and binds the E3 ligase, COP1. 12 Trib2 belongs to the pseudokinase family because it contains a central region that shares considerable homology with serine/ threonine kinases, but has deviations in the catalytic loop that are likely to eliminate catalytic activity. 13 Canonical kinases have an N-terminal lobe that binds adenosine triphosphate (ATP), a central hinge region, and a C-terminal lobe that binds substrates. 14 The N-terminal lobe contains an invariant lysine residue critical for ATP binding that is conserved in Trib2, but the highly conserved sequence, HRDLKPEN, in the C-terminal subdomain, which is critical for phosphate transfer in active kinases, is altered to LRD...

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

“…One explanation may be that the protein complexes containing TRIB2 and C/EBPα p42 or TRIB2 and p30 are different. Our previous work showed that TRIB2 binds with COP1 E3 ligase and this interaction is necessary for C/EBPα p42 degradation (13). 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.…”

“…In this study we identify the molecular mechanism involved in the dysregulation of C/EBPα expression via TRIB2 in AML. We and others (10,13,30) have previously demonstrated that TRIB2 overexpression induces AML and that it degrades C/EBPα p42. Here we provide novel insights on this process and show that the presence of C/EBPα p42 is required not only to initiate TRIB2 AML, but also for TRIB2 to cooperate with C/EBPα (p42 loss and increased p30) in driving AML disease.…”

“…In fact, TRIB2 leads to the degradation of C/EBPα p42 via E3 ligase COP1 binding whilst sparing p30 from degradation, resulting in disturbed granulopoiesis. This modulation of C/EBPα was found to be critical for the induction of AML in vivo (10,13). E2F1 cooperates with C/EBPα p30 to activate the Trib2 promoter in preleukaemic cells resulting in elevated TRIB2 expression.…”

“…TRIB2 is a potent oncogene capable of inducing fully penetrant AML in murine models, and can cooperate with other AML oncogenes to disrupt signaling pathways and transcription factors in AML disease (10)(11)(12)(13). TRIB2 expression was shown to be elevated in a cohort of AML patients with a mixed myeloid/lymphoid phenotype and a dysregulated CEBPA gene expression signature.…”

Developmental roles of tribbles protein family members

“…In the cytoplasm, Tribs direct the proteosomal degradation of key signaling proteins, including ACC1 (Qi et al, 2006), SMURF1, FoxO (Matsumoto et al, 2006) and C/ EBP Naiki et al, 2007;Selim et al, 2007;Dedhia et al, 2010;Keeshan et al, 2010;Grandinetti et al, 2011). Also in the cytoplasm, Tribs bind to inactivate LAP (Naiki et al, 2007), MKKs (Kiss-Toth et al, 2004;Wang et al, 2011), and Akt (Du et al, 2003).…”