Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal.
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.
Braem
Caroline V
CV
Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium.
Kas
Koen
K
Meyen
Eva
E
Debiec-Rychter
Maria
M
Van De Ven
Wim J M
WJ
Voz
Marianne L
ML
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; C104382:PLAG1 protein, human; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D005982:Glutathione Transferase