Supplementary Information
PTEN contains a hypothetical nuclear export signal.
The observation that germline and somatic mutations surrounding the F21
site cause nuclear localization of PTEN, prompted us to consider this region as a
potential nuclear export signal. Recent studies have revealed NLS-like regions
that can mediate nuclear localization of PTEN and that interact with the major
vault protein, a hypothetical nuclear transport protein (Chung et al., 2005).
PTEN lacks a canonical nuclear export signal, and the regions that control
nuclear export have not been identified. The consensus nuclear export signal is
defined by a short sequence of closely spaced, large hydrophobic amino acids,
usually leucine or isoleucine (L x(1-3) L x(2-4) L x L), that are recognized and bound
by CRM1/exportin, a nuclear export receptor (Fabbro & Henderson, 2003). The
sequence surrounding the F21A mutation (Figure 1) also contains closely spaced
leucines and other hydrophobic amino acids and thus could serve as an nuclear
export signal. We therefore investigated the effect of leptomycin B, a CRM1specific inhibitor, on the localization of PTEN. If nuclear export is CRM1dependent, leptomycin B treatment should induce nuclear accumulation of wildtype PTEN. HEK-293 cells and U87MG cells transiently transfected with Cterminal EGFP-tagged wild-type PTEN were treated with 10 or 20 ng/ml of
Leptomycin B for 4 hours, and localization was examined by fluorescence
microscopy. Under these conditions, leptomycin B had no significant effect on
the localization of PTEN suggesting that nuclear export is CRM1-independent
(data not shown). This result is consistent with recent observations in HeLa cells
expressing GFP-tagged PTEN (Liu et al., 2005). However, leptomycin B did
change the nuclear-cytoplasmic distribution of PTEN when cellular localization
was determined by cell fractionation in transiently transfected U87MG cells. At
concentrations of 10 and 20 ng/ml Leptomycin B led to partial loss of cytoplasmic
PTEN and an increase in nuclear PTEN (see attached figure). Analysis of tubulin (cytoplasmic marker) and Topoisomerase-I (nuclear marker) indicate that
there was no cross contamination between nuclear and cytoplasmic fractions
(see attached figure). Examination of p53, which depends on CRM1 for nuclear
export, indicated that leptomycin B treatment was effective. These data suggest
that a small fraction of PTEN may be exported from the nucleus in a CRM1dependent manner.
SUPPLEMENTAL MATERIALS AND METHODS
Leptomycin B Treatment
Leptomycin B treatment was performed in U87MG cells transiently
transfected with pcDNA3-PTEN-WT expression plasmid. 48 hours posttransfection the transfected cells were treated with the indicated amounts of
Leptomycin B (SIGMA®; refer to figure legend) for 4 hours. Cytoplasmic and
nuclear fractionation was performed using NE-PER® Nuclear and Cytoplasmic
Extraction Reagents (PIERCE) following the manufacturer’s protocol.
Immunoblot Analysis
Immunoblot analysis against HA-tagged PTEN, p53 (Santa Cruz Biotechnology,
Inc.), Topoisomerase I (TopoGEN, Inc.), and -Tubilin (1:1000, BD Biosciences
PharmingenTM) was performed at 1:1000 dilution as described in MATERIALS
AND METHODS. Incubation with secondary antibody was performed at
1:10,000 antibody dilutions were either ImmunoPure Goat Anti-Rabbit IgG (H+L)
Peroxidase Conjugated (Pierce) or ImmunoPure Goat Anti-Mouse IgG (H+L)
Peroxidase Conjugated (Pierce) in 5% non-fat dry milk/PBST for 1 hour at room
temperature. Membranes were washed twice with PBST and once with PBS
before chemiluminescent detection of antigen-antibody
SUPPLEMENTAL REFERENCES
Chung JH, Ginn-Pease ME and Eng C (2005). Phosphatase and tensin
homologue deleted on chromosome 10 (PTEN) has nuclear localization signallike sequences for nuclear import mediated by major vault protein. Cancer Res
65: 4108-16.
Fabbro M and Henderson BR (2003). Regulation of tumor suppressors by
nuclear-cytoplasmic shuttling. Exp Cell Res 282: 59-69.
Liu F, Wagner S, Campbell RB, Nickerson JA, Schiffer CA and Ross AH (2005).
PTEN enters the nucleus by diffusion. J Cell Biochem 96: 221-34.
Figure Legend-Effects of Leptomycin B on PTEN localization.U87MG cells
transiently transfected with wild-type PTEN were treated with vehicle (methanol)
or 5, 10, 20 ng/ml of Leptomycin B for 3 hours. After treatment, cytoplasmic and
nuclear fractions were prepared, and equal amounts of protein (50 g) from each
fraction were analyzed by immunoblotting with antibodies against HA-tag (HA-
PTEN), -tubulin (cytoplasmic marker) and Topoisomerase-I (nuclear marker) as
described under MATERIALS AND METHODS.