A conformational change in PBX1A is necessary for its nuclear localization.
The fly homeodomain (HD) protein EXTRADENTICLE (EXD) is dependent on a second HD protein, HOMOTHORAX (HTH), for nuclear localization. We show here that in insect cells the mammalian homolog of EXD, PBX1A, shows a similar dependence on the HTH homologs MEIS1, 2, and 3 and the MEIS-like protein PREP1. Paradoxically, removal of residues N-terminal to the PBX1A HD abolishes interactions with MEIS/PREP but allows nuclear accumulation of PBX1A. We use deletion mapping and fusion to green fluorescent protein to map two cooperative nuclear localization signals (NLSs) in the PBX HD. The results of DNA-binding assays and pull-down experiments are consistent with a model whereby the PBX N-terminus binds to the HD and masks the two NLSs. In support of the model, a mutation in the PBX HD that disrupts contact with the N-terminus leads to constitutive nuclear localization. The HD mutation also increases sensitivity to protease digestion, consistent with a change in conformation. We propose that MEIS family proteins induce a conformational change in PBX that unmasks the NLS, leading to nuclear localization and increased DNA-binding activity. Consistent with this, PBX1 is nuclear only where Meis1 is expressed in the mouse limb bud.
Saleh
M
M
McGill Cancer Centre, McGill University, Montréal, Québec, H3G 1Y6, Canada.
Huang
H
H
Green
N C
NC
Featherstone
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015342:DNA Probes / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015342:DNA Probes; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; C491412:Pknox1 protein, mouse; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C083400:PBX1 protein, human; D049452:Green Fluorescent Proteins