Nuclear localization domains in human thyroid transcription factor 2.
Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin alpha. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.
Romanelli
Maria Grazia
MG
Department of Mother and Child, Biology and Genetics, University of Verona, Strada le Grazie 8, 37134 Verona, Italy. mariagrazia.romanelli@univr.it
Tato'
Luciano
L
Lorenzi
Pamela
P
Morandi
Carlo
C
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051858:Forkhead Transcription Factors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D014162:Transfection; D028901:alpha Karyopherins / Q000378:metabolism
D024361:Amino Acids, Basic; D004268:DNA-Binding Proteins; C106894:FOXE1 protein, human; D051858:Forkhead Transcription Factors; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D012097:Repressor Proteins; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins