Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment

Ontario Cancer Institute, University Health Network, Toronto, ON, Canada M5G 1L7.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(16):6078-83. DOI: 10.1073/pnas.1119073109
Source: PubMed


Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both "MLL" and "c-Myb" binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.

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    • "Two such sequences were identified at positions 397–401 (Leu-Asp-Glu-Ala-Ile) and 415–419 (Met-His-Thr-Leu-Leu) of E2A; similar ϕ-x-x-ϕ-ϕ containing sequences were observed in AD2 of related HEB and E2–2 proteins (Figure 1). The presence of these sequences in tandem suggested that the corresponding regions in the E-proteins could bind to the KIX domain simultaneously and perhaps in a cooperative manner similar to that previously observed for p53 and FOXO3a (35,50). "
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    ABSTRACT: The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.
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    • "The binding is also phosphorylation dependent like that of pKID although phosphorylated AD1 binds preferentially to MLL site of KIX domain (61). Overall, the MLL site exhibits remarkable plasticity being able to accommodate a variety of peptides in various orientations without any substantial structural rearrangement (62). "
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    • "AMPK only phosphorylates nuclear FOXO3 thereby acting in concert with growth factor withdrawal or cellular stress signals. The exact mechanism of FOXO3 transcriptional activation by AMPK has not been completely elucidated but phosphorylation by AMPK increases the interaction with CREB-binding protein (CBP) (Wang et al., 2012) and p300 which both affect FOXO3 transcriptional activity and promoter recognition by acetylation. Phosphorylation of FOXO by AMPK has been linked to FOXO-induced autophagy (Chiacchiera and Simone, 2009), neuronal cell death (Davila et al., 2012), and muscle atrophy (Sanchez et al., 2012). "
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