An evolutionarily conserved nuclear export signal facilitates cytoplasmic localization of the Tbx5 transcription factor.
During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.
Kulisz
Andre
A
Department of Pediatrics, Northwestern University Feinberg School of Medicine, CMRC, 2300 Children's Plaza, Box #204, Chicago, IL 60614, USA.
Simon
Hans-Georg
HG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D019143:Evolution, Molecular; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D028884:Karyopherins / Q000037:antagonists & inhibitors; D008156:Luciferases / Q000032:analysis / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals / Q000235:genetics*; D017354:Point Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D000097606:Exportin 1 Protein
D005231:Fatty Acids, Unsaturated; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008156:Luciferases; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D020825:T-Box Domain Proteins; D000097606:Exportin 1 Protein; C103172:T-box transcription factor 5; C038753:leptomycin B