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Cancer Research 66, 11677, December 15, 2006. doi: 10.1158/0008-5472.CAN-06-2240
© 2006 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Nuclear Localization of PTEN Is Regulated by Ca2+ through a Tyrosil Phosphorylation–Independent Conformational Modification in Major Vault Protein

Takeo Minaguchi1,2, Kristin A. Waite1,2 and Charis Eng1,2,3,4,5

1 Genomic Medicine Institute, 2 Lerner Research Institute, and 3 Taussig Cancer Center, Cleveland Clinic Foundation; and 4 Department of Genetics and 5 CASE Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio

Requests for reprints: Charis Eng, Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Mailstop NE-50, Cleveland, OH 44195. Phone: 216-444-3440; Fax: 216-636-0655; E-mail: engc{at}ccf.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown in MCF-7 cells that nuclear phosphatase and tensin homologue deleted on chromosome 10 (PTEN) down-regulates phosphorylation of p44/42 and cyclin D1 and induces G1 cell cycle arrest, whereas cytoplasmic PTEN down-regulates phosphorylation of Akt, up-regulates p27, and induces apoptosis. In this manner, nucleocytoplasmic partitioning of PTEN seems to differentially regulate the cell cycle and apoptosis. We have also reported that PTEN has nuclear localization signal–like sequences required for major vault protein (MVP)–mediated nuclear translocation. To date, several other proteins are reported to interact with MVP, including extracellular signal-regulated kinases and steroid receptors, suggesting that MVP is likely to be involved in signal transduction through nucleocytoplasmic transport. However, the exact mechanism of MVP-mediated nucleocytoplasmic shuttling remains elusive. PTEN reportedly interacts in vitro with the EF hand–like motif of MVP in a Ca2+-dependent manner. The current study shows that small interfering RNA–mediated MVP silencing decreases the nuclear localization of PTEN and increases phosphorylation of nuclear p44/42. We show in situ that PTEN-MVP interaction is Ca2+ dependent and is abolished by Mg2+. Nuclear localization of PTEN is decreased by increasing Ca2+ levels in culture medium in a dose-dependent manner. Ca2+ ionophore A23187 increases nuclear localization of PTEN and decreases phosphorylation of nuclear p44/42. Finally, we show that Ca2+-dependent PTEN-MVP interaction is not related to MVP's tyrosil phosphorylation but rather due to its conformational modification. Our observations suggest that Ca2+ regulates PTEN's nuclear entry through a tyrosil phosphorylation–independent conformational change in MVP. Collectively, our data present evidence of a novel crosstalk between the Ca2+ signaling–mediated regulation of the cell cycle and MVP-mediated nuclear PTEN localization and function. (Cancer Res 2006; 66(24): 11677-82)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Somatic alterations of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) are common events in diverse human cancers, including carcinomas of the breast, endometrium, prostate, and glioblastoma. Germ line mutations of PTEN predispose to dominantly inherited Cowden and Bannayan-Riley-Ruvalcaba syndromes, which are characterized by the formation of multiple benign tumors and an increased risk of malignant and benign breast, thyroid, and endometrial tumors (1). PTEN protein is composed of an NH2-terminal phosphatase domain containing the consensus phosphatase motif, a C2 domain that binds to phospholipid membranes, and a COOH-terminal tail containing a PDZ-binding domain (2, 3). PTEN is a dual-specificity phosphatase: PTEN's protein phosphatase activity (PPA) dephosphorylates tyrosine-, serine-, and threonine-phosphorylated peptides in vitro and dephosphorylates tyrosine in focal adhesion kinase in vivo; PTEN's lipid phosphatase activity dephosphorylates phosphatidylinositol-(3,4,5)-triphosphate, a lipid second messenger and a regulator of the phosphatidylinositol 3-kinase/Akt pathway (1). PTEN's PPA also regulates the mitogen-activated protein kinase (MAPK) pathway (4). The many important cellular functions of PTEN include a wide range of biological processes (i.e., G1 cell cycle arrest, apoptosis, cell migration, spreading, polarity, chemotaxis, and focal adhesion formation; refs. 1, 5).

Several known tumor suppressors have been shown to undergo nuclear-cytoplasmic shuttling, such as APC, BRCA1, NF2, p53, and p27. In addition to these, immunohistochemical analysis of PTEN revealed nuclear PTEN expression in several tissue types, which seemed to be highest in normal cells and diminish, with concomitant increase in cytoplasmic expression, with neoplastic progression (6). Our previous study in MCF-7 cells showed that nuclear PTEN protein levels peak during G0-G1 phases and nadir during S phase of the cell cycle (7). We have further shown that nuclear PTEN down-regulates phosphorylation of MAPK, down-regulates cyclin D1, and induces G1 cell cycle arrest, whereas cytoplasmic PTEN down-regulates phosphorylation of Akt, up-regulates p27, and induces apoptosis (8). These observations suggest that nuclear-cytoplasmic partitioning of PTEN differentially regulates the cell cycle and apoptosis and provide further evidence that nuclear import of PTEN should play a role in carcinogenesis. In this context, the mechanism of nucleocytoplasmic shuttling of PTEN is thought to be crucial for the regulation of nuclear and cytoplasmic functions of PTEN.

The vault complex is an evolutionarily conserved ribonucleoprotein particle with a molecular mass of 13 MDa and is hypothesized as a general carrier molecule for nuclear-cytoplasmic transport (9). The vault is composed of multiple copies of three proteins [major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase-associated protein 1] and small untranslated RNA molecules. The main component is MVP, which constitutes over 70% of the total mass of the vault and determines its structure. To date, several proteins have been reported to interact with MVP, including PTEN (10); estrogen, progesterone, and glucocorticoid receptors (11); extracellular signal-regulated kinases (Erk; ref. 12); and constitutively photomorphogenic 1 (COP1), which is an inhibitor of activator protein-1 (AP-1) transcription (13). Thus, MVP is likely to be involved in signal transduction pathways through nucleocytoplasmic transport of the cargo proteins. However, the molecular mechanism of MVP-mediated nucleocytoplasmic shuttling remains to be elucidated.

We have recently shown that PTEN has bipartite nuclear localization signal (NLS)–like sequences required for MVP-mediated nuclear import of PTEN (14). MVP reportedly interacts with the C2 domain of PTEN through the EF hand–like motif in a Ca2+-dependent manner in HeLa cells (10). Phosphorylation of MVP depends on the presence of Mg2+ in PC12 cells (15), and epidermal growth factor (EGF) stimulation increases tyrosil phosphorylation of MVP in WI38 cells (12). Therefore, we hypothesized that Ca2+, Mg2+, and tyrosil phosphorylation of MVP may be playing crucial roles in MVP function, particularly in nuclear import of PTEN. To elucidate the mechanism of MVP-mediated nuclear transport of PTEN, we examined the effects of Ca2+ and Mg2+ on the interaction between PTEN and MVP, subcellular localization of PTEN, and tyrosil phosphorylation and potential conformational changes of MVP in the breast cancer cell line MCF-7. Our findings provide novel insights into the mechanism of MVP-mediated nucleocytoplasmic trafficking and also of the regulation of the nuclear and cytoplasmic functions of PTEN through subcellular compartmentalization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. MCF-7 breast cancer cells were cultured in high-glucose DMEM containing 10% fetal bovine serum (FBS), penicillin, and streptomycin. The MCF-7 Tet-Off cell line expressing wild-type PTEN (PTEN:WT) was generated and maintained as previously described (14). Vector expression was controlled with 2 µg/mL tetracycline (Tet). Stock solutions of Ca2+ ionophore A23187 (Calbiochem, San Diego, CA) were prepared in 99.7% DMSO and were added to the culture medium as specified in the text.

Small interfering RNA assay. The following small interfering RNA (siRNA) sequences were synthesized (Integrated DNA Technologies, Coralville, IA): MVP siRNA (plus strand, 5'-GUUUGAGGAGGUUCUGGAUTT-3'; minus strand, 5'-AUCCAGAACCUCCUCAAACTT-3') and scrambled siRNA (plus strand, 5'-AUUAGGUGAUUUGGUGGGCTT-3'; minus strand, 5'-GCCCACCAAAUCACCUAAUTT-3'). MCF-7 cells were plated at 30% to 40% confluence in six-well plates and were transfected the following day with 100 pmol/well of MVP or scrambled siRNA using Lipofectamine 2000 (Invitrogen, San Diego, CA) in serum-free medium. Twenty-four hours after transfection, cells were placed in complete medium containing 10% FBS and incubated for additional 48 hours before harvesting.

Plasmid construction and transfection. To construct pcDNA3.1D/V5-His/MVP, the coding region of MVP was amplified by reverse transcription-PCR using cDNA isolated from MCF-7 cells as a template with the following primers: 5'-CACCATGGCAACTGAAGAGTTC-3' and 5'-GCGCAGTACAGGCACCACGT-3'. The 2.7-kb PCR product of MVP was directionally cloned into pcDNA3.1D/V5-His-TOPO vector (Invitrogen), which contains a V5 and poly-histidine epitope tag at the COOH terminus, according to the manufacturer's protocol. The PCR product and the construct were confirmed by DNA sequencing. The generated construct was transiently transfected into MCF-7 or PTEN:WT cells using Lipofectamine 2000 (Invitrogen) in antibiotics-free, serum-free medium for 24 hours as recommended by the manufacturer's instructions and incubated in complete medium for additional 24 hours before harvesting.

SDS-PAGE, immunoblotting, and antibodies. Whole cell–free protein extracts were prepared according to the manufacturer's recommendations (M-Per, Pierce Biotechnology, Rockford, IL). Nuclear and cytoplasmic proteins were isolated with a buffer extraction system and centrifugation according to the manufacturer's recommendations (NE-Per, Pierce Biotechnology). Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting. The monoclonal antibody 6H2.1 raised against the last 100 COOH-terminal amino acids of PTEN (Cascade Biosciences, Portland, OR) was used for PTEN immunoblotting and immunoprecipitation (16). Monoclonal anti-MVP (Lab Vision, Fremont, CA), monoclonal anti-V5 (Invitrogen), monoclonal anti–phospho-tyrosine (Cell Signaling Technology, Beverly, MA), monoclonal anti-actin (Santa Cruz, Santa Cruz, CA), anti–phospho-p44/42, and monoclonal anti-p44/42 (Cell Signaling Technology, Danvers, MA) antibodies were used.

Immunoprecipitation. Cells were harvested, washed with PBS, and sonicated in lysis buffer [50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 1% NP40] containing inhibitors. After pretreatment with protein A/G-Sepharose beads (Santa Cruz) and normal mouse IgG (Santa Cruz), cell lysates (800–1,000 µg protein) were incubated with the respective antibodies overnight at 4°C and then incubated for 3 hours with protein A/G-Sepharose. The immune complexes were isolated via centrifugation and washed four times in TNN buffer [20 mmol/L Tris-HCl (pH 8), 100 mmol/L NaCl, 0.5% NP40] before boiling in SDS sample buffer and separation by 8% SDS-PAGE.

Protease sensitivity assay. Protease sensitivity assay was done as described elsewhere (17). Briefly, immunoprecipitations of recombinant MVP were prepared with anti-V5-tag antibody from transiently transfected MCF-7 cells, and the beads were washed with TNN buffer containing Ca2+ and/or Mg2+, split to the same volume, and incubated overnight at 37°C with 0, 5, 50, or 500 ng of sequencing grade trypsin in the presence of the same concentration of Ca2+ and/or Mg2+ as lysis buffer and TNN buffer. Samples were separated by 8% SDS-PAGE and immunoblotted with anti-MVP antibody.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
siRNA-mediated silencing of MVP decreases the nuclear localization of PTEN and increases phosphorylation of nuclear p44/42 MAPK. We have previously reported that PTEN has NLS-like sequences required for interaction with MVP and nuclear localization of PTEN (14). To further confirm the role of MVP in PTEN nuclear import, we examined the effect of MVP silencing by siRNA on subcellular localization of PTEN. MVP-targeted siRNA significantly decreased total MVP protein level compared with the scrambled siRNA-treated control (Fig. 1A ). As expected, the nuclear localization of PTEN was significantly decreased in cells treated with MVP siRNA compared with the control (Fig. 1A and B). Moreover, nuclear p44/42 was significantly phosphorylated by MVP siRNA, whereas cytoplasmic p44/42 was unchanged (Fig. 1A and C). These findings confirm that MVP is involved in the nuclear localization of PTEN and also suggest that MVP is regulating downstream functions of nuclear PTEN through nuclear transport.


Figure 1
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  		Figure 1. siRNA-mediated silencing of MVP decreases nuclear localization of PTEN and increases phosphorylation of nuclear p44/42 MAPK. A, MCF-7 cells were transfected with MVP-targeted siRNA (MVP) or scrambled siRNA (Cont) in serum-free medium for 24 hours and incubated in complete medium for an additional 48 hours before harvesting. Protein levels of MVP, PTEN, phospho-p44/42 (P-p44/42), p44/42, and actin were examined by immunoblotting in whole-cell extracts and nuclear-cytoplasmic fractions. B, data in (A) were quantified by densitometric analysis, and each ratio of nuclear to cytoplasmic fraction was calculated and normalized to control. C, each ratio of phospho-p44/42 to p44/42 was calculated and normalized to control. Columns, mean (n = 3); bars, SD.

 
Interaction between PTEN and MVP in situ is Ca2+ dependent and abolished by Mg2+. It has been reported that the interaction between PTEN and MVP depends on the presence of Ca2+ in glutathione S-transferase pull-down assay (10). Because phosphorylation of MVP is reportedly dependent on the presence of Mg2+ (15), and because Ca2+ and Mg2+ are known to exert opposing effects in many cellular functions (18, 19), we hypothesized that Ca2+ and Mg2+ might be involved in the interaction between PTEN and MVP. Immunoprecipitation showed that Ca2+-dependent interaction of PTEN with MVP was abolished by addition of the same concentration of Mg2+ as Ca2+ (Fig. 2A ). Because the effect of Ca2+ on the interaction between PTEN and MVP is reported to be dose dependent (10), we subsequently examined a dose-effect of Mg2+ on the interaction. Interestingly, as Mg2+ concentration increases, the PTEN-MVP interaction slightly increases at lower concentrations of Mg2+ and then is abolished at the half concentration of Ca2+ (Fig. 2B). These findings indicate that Mg2+ antagonizes Ca2+-dependent interaction of PTEN with MVP, and the antagonizing effect of Mg2+ is at least 2-fold stronger than Ca2+ at the same concentration.


Figure 2
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  		Figure 2. Mg2+ abolishes Ca2+-dependent interactions between PTEN and MVP. PTEN:WT cells were incubated for 48 hours in Tet-free medium before harvesting, and the PTEN-MVP interaction was examined by immunoprecipitation (IP). A, MVP was precipitated with anti-PTEN antibody in the presence of 10 mmol/L CaCl2 and/or 10 mmol/L MgCl2 and immunoblotted (IB) with anti-PTEN or anti-MVP antibody. For control, immunoprecipitation was done in the absence of CaCl2 and MgCl2. B, MVP was precipitated with anti-PTEN antibody in the presence of the indicated concentrations of CaCl2 and MgCl2.

 
Nuclear localization of PTEN is decreased by increasing Ca2+ concentrations in the culture medium in a dose-dependent manner. Because the interaction between PTEN and MVP is reportedly affected by Ca2+ in a dose-dependent manner (10), we next examined a dose-effect of extracellular Ca2+ on nuclear localization of PTEN. MCF-7 cells were subjected to artificial concentrations of Ca2+ via the addition of CaCl2 in the culture medium. Incubation in a dose-gradient increase of Ca2+ showed a dose-dependent decrease of nuclear localization of PTEN (Fig. 3A and B ). This finding suggests that cellular Ca2+ concentration may be regulating nuclear localization of PTEN.


Figure 3
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  		Figure 3. Nuclear localization of PTEN is decreased by increasing Ca2+ concentration in the culture medium in a dose-dependent manner. A, After 24 hours of serum deprivation, MCF-7 cells were incubated in complete media supplemented with CaCl2 for an additional 24 hours. Calculated concentrations of Ca2+ in the medium are indicated. Protein levels of PTEN and actin were examined by immunoblotting in whole-cell extracts and nuclear-cytoplasmic fractions. B, data in (A) were quantified by densitometric analysis, and ratios of PTEN's nuclear-to-cytoplasmic fractions were calculated and normalized to the ratio of 1.6 mmol/L Ca2+. Columns, mean (n = 3); bars, SD.

 
Calcium ionophore A23187 increases nuclear localization of PTEN and decreases phosphorylation of nuclear p44/42 MAPK in a dose-dependent manner. A23187 is known to increase cytoplasmic Ca2+ levels by releasing it from the endoplasmic reticulum Ca2+ store. To confirm the regulation of nuclear PTEN localization by Ca2+, we examined the effect of A23187 on PTEN's localization. Incubation with 0.1 or 1.0 µmol/L A23187 increased nuclear localization of PTEN (Fig. 4A and B ). To determine if the PTEN that was translocated to the nucleus was active, we further examined the downstream effects of this increased nuclear PTEN. A23187 incubation decreased phosphorylation of nuclear p44/42 in a dose-dependent manner (Fig. 4A and C). These data suggest that intracellular increases of Ca2+ up-regulates nuclear localization of PTEN, which consequently down-regulates phosphorylation of nuclear p44/42.


Figure 4
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  		Figure 4. Calcium ionophore A23187 increases nuclear localization of PTEN and decreases phosphorylation of nuclear p44/42 MAPK in a dose-dependent manner. A, after 24 hours of serum deprivation, MCF-7 cells were incubated in complete medium with 0.01, 0.1, or 1.0 µmol/L A23187 for an additional 24 hours before harvesting. As a control, cells were incubated in the complete medium added with appropriate volumes of DMSO. Protein levels of PTEN, actin, p44/42, and phospho-p44/42 were examined by immunoblotting in whole-cell extracts and nuclear-cytoplasmic fractions. B, data in (A) were quantified by densitometric analysis, and each ratio of PTEN's nuclear to cytoplasmic fraction was calculated and normalized to the ratio of DMSO 10–5%. C, each ratio of nuclear phospho-p44/42 to p44/42 of A23187 was divided by corresponding ratio of DMSO and normalized to the value of 0.01 µmol/L A23187. Columns, mean (n = 3); bars, SD.

 
Ca2+-dependent PTEN-MVP interaction is not regulated by tyrosil phosphorylation of MVP. Phosphorylation of MVP is reported to depend on the presence of Mg2+ (15), and EGF stimulation increases MVP tyrosil phosphorylation (12). Additionally, we have recently found that subcellular localization of PTEN is not regulated by PTEN phosphorylation (14). Therefore, we hypothesized that Ca2+ and Mg2+ might be regulating PTEN-MVP interaction and PTEN nuclear transport through tyrosil phosphorylation of MVP. We first verified the interaction of recombinant MVP with PTEN in situ by immunoprecipitation. Recombinant MVP interacted with PTEN in the presence of Ca2+ and addition of Mg2+ abolished the interaction (Fig. 5 ), which is consistent with the result on endogenous MVP (Fig. 2A). Next, we examined the effect of Ca2+ and/or Mg2+ on tyrosil phosphorylation of MVP. No significant differences were found in tyrosil phosphorylation of MVP among Ca2+, Ca2+ plus Mg2+, Mg2+, and control samples (Fig. 5A and B). These observations suggest that MVP's Ca2+-dependent interaction with PTEN is not regulated by MVP tyrosil phosphorylation.


Figure 5
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  		Figure 5. Ca2+- and Mg2+-dependent PTEN-MVP interaction is not related to tyrosil phosphorylation of MVP. A, to first validate the interaction of recombinant MVP with PTEN, PTEN:WT cells were transiently transfected with an MVP construct in Tet-free medium for 48 hours before harvesting. Recombinant MVP was precipitated with anti-PTEN antibody in the presence of 10 mmol/L CaCl2 and/or 10 mmol/L MgCl2 as indicated and immunoblotted with anti-V5-tag or anti-PTEN antibody. For the evaluation of MVP's tyrosil phosphorylation, MCF-7 cells were transiently transfected with the MVP construct and harvested 48 hours after transfection. Recombinant MVP was precipitated with anti-V5-tag antibody in the presence of 10 mmol/L CaCl2 and/or 10 mmol/L MgCl2 as indicated and immunoblotted with anti–phospho-tyrosine (p-Tyr) or anti-MVP antibody. For control, immunoprecipitation was done in the absence of CaCl2 and MgCl2. B, data in (A) were quantified by densitometric analysis, and ratios of phospho-tyrosine to MVP were calculated and normalized to control. Columns, mean (n = 3); bars, SD.

 
Ca2+ causes a conformational modification in MVP. It is known that Ca2+ may result in conformational changes of proteins containing the Ca2+-binding EF hand motif (20). Hence, we next examined the effect of Ca2+ and Mg2+ on the conformational change of recombinant MVP by partial tryptic digestion. Digestions with 5 and 50 ng trypsin in the presence of Ca2+ shows an increase of larger fragments from recombinant MVP compared with Ca2+/Mg2+-free control, Ca2+ plus Mg2+, or Mg2+ samples, indicating that Ca2+ decreases recombinant MVP's sensitivity to trypsin, and Mg2+ antagonizes the effect (Fig. 6 ). This finding indicates that Ca2+ causes MVP's conformational modification, and Mg2+ antagonizes it.


Figure 6
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  		Figure 6. Ca2+ decreases recombinant MVP's sensitivity to tryptic digestion, and Mg2+ antagonizes the effect. MCF-7 cells were transiently transfected with MVP construct and harvested 48 hours after transfection. Recombinant MVP was precipitated with anti-V5-tag antibody in the presence of CaCl2 and/or MgCl2 as indicated, washed, split to the same volume, and partially digested at 37°C overnight with 0, 5, 50, or 500 ng trypsin. Samples were separated by 8% SDS-PAGE and immunoblotted with anti-MVP antibody. Arrows, digestions in the presence of Ca2+ alone showed an increase of larger fragments from recombinant MVP after 5 and 50 ng tryptic digestion compared with the other samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that PTEN has four putative NLS-like sequences, and that two double-site mutations cause nuclear localization defects of PTEN and abolish the interaction with MVP, concluding that MVP mediates PTEN nuclear import (14). However, another group has recently reported that PTEN's nuclear entry is mediated by passive diffusion (21). To further confirm our conclusion, the present study examined the effect of siRNA-mediated silencing of MVP on PTEN subcellular localization. As expected, MVP-targeted siRNA significantly decreased total protein level of MVP and nuclear localization of PTEN compared with the control (Fig. 1A and B). Although possibilities of other pathways in PTEN nuclear import, such as passive diffusion and mediation by other transporters, still cannot be excluded, our data suggest that MVP-mediated nuclear import is a major mechanism of PTEN translocation because we were able to significantly diminish PTEN nuclear localization in MVP siRNA-treated cells. Additionally, we have recently reported that PTEN in both wild-type PTEN-overexpressing MCF-7 cells as well as in PTEN nuclear localization defect cells interacts equally with importin-{alpha} and importin-ß (14). Therefore, it is possible that there are NLS-like sequence-independent pathways of PTEN nuclear import mediated by importins as well as passive diffusion. Although further work is required to clarify this issue, multiple pathways are likely to be involved in the nuclear import of PTEN, as analogously with Erk. Erk has been shown to be imported to the nucleus by two coexisting mechanisms: passive diffusion and Ran-dependent active transport (22). Interestingly, MVP is reported to interact with phosphorylated p44/42 (Erks) in response to EGF stimulation (12). If MVP is involved in nuclear import of phospho-p44/42 as well, MVP-targeted siRNA should decrease nuclear phospho-p44/42. However, our results show that nuclear localization of phospho-p44/42 is increased, most likely due to the upstream event (i.e., decreased nuclear localization of PTEN; Fig. 1B).

Because published findings suggest that Ca2+ and Mg2+ play important roles in MVP's function (10, 12), and because in many cellular processes these divalent cations are known to act antagonistically towards each other (18, 19), we hypothesized that Ca2+ and Mg2+ might be regulating the PTEN-MVP interaction by antagonizing each other such that they regulate nuclear import of PTEN. Our immunoprecipitation results show that Mg2+ antagonizes Ca2+-dependent interaction between PTEN and MVP (Fig. 2A and B). Therefore, we speculate that nuclear localization of PTEN is regulated by subtle differences of Ca2+ and Mg2+ concentrations between the cytoplasm and nucleus by affecting the interaction with MVP. We further examined the effect of increased cytoplasmic Ca2+ levels on PTEN nuclear localization by incubation with the calcium ionophore A23187. As expected, A23187 increased nuclear localization of PTEN (Fig. 4A and B). It can be speculated that the increased PTEN-MVP interaction due to cytoplasmic Ca2+ accumulation results in increased import of PTEN proteins to the nucleus. However, the result of our experiment manipulating extracellular Ca2+ (i.e., in the culture medium) was apparently contradictory to the results obtained with A23187 (Fig. 3A and B). The mechanism whereby extracellular increases of Ca2+ levels decrease the nuclear localization of PTEN may be explained by PTEN being unable to dissociate from MVP even in the nucleus due to an increased interaction intensity because of the steep increase in nuclear Ca2+. Additionally, the results of our experiments using A23187 suggest that increased nuclear localization of PTEN consequently down-regulates phosphorylation of p44/42 MAPK (Fig. 4A and C). This downstream effect is consistent with our previous observations in PTEN's nuclear localization defect mutants that nuclear PTEN down-regulates phosphorylation of p44/42 and cyclin D1 and induces G0-G1 cell cycle arrest (8). Combined with these recent findings, our data are suggestive of the possibility that nuclear PTEN induces G0-G1 cell cycle arrest by Ca2+ signaling–regulated nuclear import. Ca2+ signaling is known to regulate G1-S progression of the cell cycle through promoting phospholipase C/protein kinase C/MAPK and calmodulin (CaM)/CaM-dependent kinase (CaM-K) kinase/CaM-K pathways (23–26). Therefore, it is reasonable to postulate that Ca2+ signaling is also regulating nuclear entry of PTEN, resulting in modulation of G1 cell cycle arrest. However, it is necessary to further elucidate how calcium signaling up-regulates and down-regulates G1 cell cycle arrest through nuclear PTEN-dependent and PTEN-independent pathways.

Recently, it has been reported that UV irradiation enhances tyrosil phosphorylation of MVP and causes dissociation of COP1, which is an inhibitor of AP-1 transcription, from MVP (13). Additionally, the interaction between MVP and Src tyrosine kinase is also dependent on tyrosil phosphorylation of MVP (27). Because EGF stimulation increases tyrosil phosphorylation of MVP (12, 27), and because we previously found that PTEN's phosphorylation status is not correlated with its subcellular localization (14), we hypothesized that Ca2+/Mg2+–dependent MVP-PTEN interactions may be mediated by tyrosil phosphorylation of MVP. However, interestingly, our data suggest that the Ca2+/Mg2+–dependent interaction of MVP with PTEN is not regulated by MVP's tyrosil phosphorylation (Fig. 5). Nevertheless, tyrosil phosphorylation of MVP is likely to play an important role in the growth factor/receptor tyrosine kinase/MAPK pathway because tyrosil phosphorylated MVP reportedly forms a complex with SHP-2 and Erks in response to EGF stimulation (12). Besides, it has been recently shown that EGF stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src, and that the EGF-dependent MVP-Src interaction down-regulates phosphorylation of MAPK (27). Hence, there seems to be tyrosil phosphorylation–dependent and tyrosil phosphorylation–independent mechanisms regulating MVP-mediated MAPK pathway.

Because our data showed that tyrosil phosphorylation of MVP did not mediate ion-associated MVP-PTEN interactions, we pursued other mechanisms. Ca2+ binding to EF hand motifs is known to cause conformational changes within many proteins containing EF hand motifs, including calmodulin, neuronal calcium sensor family, and S100 protein family (20). An EF hand–like motif of MVP interacts with C2 domain of PTEN in a Ca2+-dependent manner (10). Because PTEN's C2 domain has a Ca2+-independent motif (28), we examined the effect of the interplay of Ca2+/Mg2+ on recombinant MVP's conformational change. Our results here indicate that Ca2+ induces a conformational change within MVP, and Mg2+ antagonizes this effect (Fig. 6). We therefore postulate that Ca2+ binding to MVP causes MVP's conformational alteration so that the affinity of MVP's EF hand–like motif for PTEN's C2 domain is enhanced, and that Mg2+ has a competitive effect on Ca2+ binding to MVP, although our results suggest that Mg2+ has agonistic effects at a lower Mg2+/Ca2+ ratio as well (Fig. 2B). We believe our postulate based on our data (i.e., Ca2+-dependent regulation of nuclear entry by MVP) is plausible, as Ca2+ is also reported to regulate opening and closing of the nuclear pore complex through its conformation change (29).

In summary, we show here that Ca2+ is regulating the nuclear localization of PTEN and its downstream effect (i.e., phosphorylation of nuclear p44/42), by modulating the interaction with MVP, and that the Ca2+-dependent PTEN-MVP interaction is attributable to MVP's conformational change but not to MVP's tyrosil phosphorylation. Importantly, our findings provide a novel crosstalk pathway between MVP-mediated nuclear transport and function of PTEN and the Ca2+ signaling–mediated regulation of the cell cycle in breast cancer.


   Acknowledgments
 
Grant support: American Cancer Society grant RSG02-151-01CCE (C. Eng) and Doris Duke Distinguished Clinical Scientist Award (C. Eng).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

T. Minaguchi thanks many members of the Eng lab for helpful discussions and technical advice.


   Footnotes
 
Note: C. Eng is an honorary fellow of Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge, United Kingdom.

Received 6/19/06. Revised 9/24/06. Accepted 10/13/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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