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J. Biol. Chem., Vol. 277, Issue 22, 19673-19678, May 31, 2002
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From the Laboratory for Molecular Oncology, Department of Human
Genetics, University of Leuven and Flanders Interuniversity
Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium
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
Identification of a Karyopherin
2 Recognition Site in PLAG1,
Which Functions As a Nuclear Localization Signal*
,
2 as a PLAG1-interacting protein. Physical
interaction between PLAG1 and karyopherin 2 was confirmed by an
in vitro glutathione S-transferase
pull-down assay. Karyopherin 2 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 2 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 
-galactosidase. In
contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in
karyopherin 2 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 2 recognition site.
*
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 L
lrente
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.
§
Postdoctoral Fellow of the FWO.
¶
To whom correspondence should be addressed. Tel.:
32-016-34-59-87; Fax: 32-016-34-60-73; E-mail:
wim.vandeven@med.kuleuven.ac.be.
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