title: Critical role for a single leucine residue in leukemia induction by E2A-PBX1.
abstract: In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.
authors.affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Bayly
authors.forename: Takayuki
authors.initials: T
authors.lastname: Murase
authors.forename: Brandy D
authors.initials: BD
authors.lastname: Hyndman
authors.forename: Rachel
authors.initials: R
authors.lastname: Savage
authors.forename: Salima
authors.initials: S
authors.lastname: Nurmohamed
authors.forename: Kim
authors.initials: K
authors.lastname: Munro
authors.forename: Richard
authors.initials: R
authors.lastname: Casselman
authors.forename: Steven P
authors.initials: SP
authors.lastname: Smith
authors.forename: David P
authors.initials: DP
authors.lastname: LeBrun
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D005260:Female; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D007938:Leukemia / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000166:cytology; D041681:NIH 3T3 Cells; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D018398:Homeodomain Proteins; D015514:Oncogene Proteins, Fusion; C089847:E2A-Pbx1 fusion protein; D050882:CREB-Binding Protein; D007930:Leucine