Binding Sites; Cell Nucleus/metabolism; DNA-Binding Proteins/chemistry/metabolism; Glutathione Transferase/metabolism; Humans; Microscopy, Fluorescence; Plasmids/metabolism; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins/metabolism; Two-Hybrid System Techniques; alpha Karyopherins/chemistry/metabolism
Abstract :
[en] The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Braem, Caroline V
Kas, Koen
Meyen, Eva
Debiec-Rychter, Maria
Van De Ven, Wim J M
Voz, Marianne ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Language :
English
Title :
Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal.
Publication date :
2002
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
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