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 CRM1-specific inhibitor, on the localization of PTEN. If nuclear export is CRM1-dependent, leptomycin B treatment should induce nuclear accumulation of wild-type PTEN. HEK-293 cells and U87MG cells transiently transfected with C-terminal 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 b-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 CRM1-dependent 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 post-transfection 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 b-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 signal-like 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 mg) from each fraction were analyzed by immunoblotting with antibodies against HA-tag (HA-PTEN), b-tubulin (cytoplasmic marker) and Topoisomerase-I (nuclear marker) as described under MATERIALS AND METHODS.