MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
Identification of a Karyopherin α2 Recognition Site in PLAG1, Which Functions As a Nuclear Localization Signal*

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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 α2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin α2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin α2 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 α2 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 β-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin α2 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 α2 recognition site.

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Published, JBC Papers in Press, March 6, 2002, DOI 10.1074/jbc.M112112200

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This work was supported in part by the Geconcerteerde Onderzoeksactie (GOA, 1997–2001), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and the Algemene Spaar en Lÿlrente Kas-programma voor Kankeronderzoek. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

An “Aspirant” of the FWO.

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Postdoctoral Fellow of the FWO.