Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tolkacheva, T.
Right arrow Articles by Chan, A. M-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tolkacheva, T.
Right arrow Articles by Chan, A. M-L.
[Cancer Research 61, 4985-4989, July 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Regulation of PTEN Binding to MAGI-2 by Two Putative Phosphorylation Sites at Threonine 382 and 3831

Tatyana Tolkacheva, Manoranjan Boddapati, Anthony Sanfiz, Kunihiro Tsuchida, Alec C. Kimmelman and Andrew M-L. Chan2

The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029 [T. T., M. B., A. S., A. C. K., A. M. C.], and the Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan [K. T.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussions
 REFERENCES
 
We have reported previously that the PTEN COOH-terminal 33 amino acids play a role in the maintenance of PTEN protein stability (Tolkacheva and Chan, Oncogene, 19: 680–689, 2000). By site-directed mutagenesis, we identified two threonine residues within this COOH-terminal region at codon 382 and 383 that may be targets for phosphorylation events. Interestingly, PTEN mutants rendered phosphorylation-incompetent at these two sites, T382A/T383A, and were found to have drastically reduced expression in cultured cells. The enhanced degradation of PTEN was most likely mediated by the proteosome-dependent pathway, we have evidence that PTEN was polyubiquitinated. More interestingly, the non-phosphorylated forms of PTEN displayed significantly greater binding affinity than the wild-type protein to a previously identified PTEN interacting partner, MAGI-2/ARIP1. On the basis of all these data, we propose that PTEN recruitment to the cell-cell junction may be regulated through the phosphorylation of its COOH terminus.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussions
 REFERENCES
 
PTEN is a bona fide human tumor suppressor gene which encodes a protein with phosphatase activity toward PI3 (1, 2, 3) . The frequent inactivation of PTEN in various human tumors provides strong evidence that uncontrolled PI turnover plays a role in tumor formation (4) . Indeed, components of the PI-dependent signaling cascade, such as growth factor receptors, Ras, Akt, and PI3-K have been shown to be either constitutively activated or overexpressed in human cancer (5) . The ectopic expression of PTEN in various tumor cell lines has been shown to exert inhibitory effects on several known biological actions of PI3-K, such as proliferation, invasion, and cell survival (6) .

Biochemically, PTEN mediates the dephosphorylation of the D3-phosphate of PIP3 (7, 8, 9) . On the basis of the primary amino acid sequence and the recently resolved crystal structure (10) , PTEN can be generally divided into two functional regions. In the NH2-terminal half of the molecule resides the phosphatase and Tensin-homology domains which binds and catalyzes the dephosphorylation of phospholipid substrates. The COOH-terminal half of PTEN is most likely regulatory in nature and is composed of three sub-domains: (a) a C2 domain that has been implicated in phospholipid-binding; (b) two tandem PEST domains that are believed to play a role in controlling protein stability; and (c) a PDZ-binding domain which may play a role in protein-protein interaction.

Insights into the regulation of PTEN phosphatase activity were provided by the identification of binding proteins that interact with the COOH-terminal PDZ-binding consensus sequence of PTEN. By screening yeast two-hybrid libraries with PTEN as bait, three separate groups have identified members of the MAGUK family, hdlg, hMAST205, ARIP1/AIP1/MAGI-2 (hereafter referred to as MAGI-2), and MAGI-3, to interact directly with PTEN (11, 12, 13) . Common to all these proteins are the possession of five to six PDZ domains, two WW domains and a guanylate kinase-like domain. Immunocytochemical analysis of MAGUK proteins has shown them to be localized to cell-cell junctions. Also, expression of MAGI-2 enhances the ability of PTEN to down-regulate the PI3-K-dependent signaling cascade (12) . These results lead to the speculation that MAGUK proteins, by recruiting PTEN to the plasma membrane, promote the dephosphorylation of PIP3 in that cellular compartment. However, it is unclear how the interaction between PTEN and MAGUK family proteins is being regulated.

In this study, we report the identification of two potential phosphorylation sites in the PTEN COOH terminus that may play a role in controlling the binding affinity to the second PDZ domain (PDZ2) of MAGI-2. Our results provide a model whereby PTEN localization and activity in a cell could be regulated through phosphorylation.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussions
 REFERENCES
 
Cell Culture.
The NIH3T3 cell line was maintained in DMEM supplemented with 10% calf serum. The 293T and PC3 cell lines were maintained in DMEM supplemented with 10% FCS. For transfecting 293T cells, the standard calcium phosphate precipitation method was used. For gene transfer in PC3 cells, ~106 cells on a 60-mm culture dish were lipofected with 1–2 µg of DNA using 8 µl of Lipofectamine (Life Technologies, Inc., Rockville, MD) in 1.4 ml of serum- and antibiotic-free DMEM. After 5 h of incubation at 37°C, cells were then rinsed once and replaced with 3 ml of growth medium. Most biological assays were performed within 36–48 h after transfection.

Plasmids.
PTEN-WT, PTEN-AA, and various PTEN COOH-terminal truncated cDNAs have been described previously (14) . For the generation of PTEN T382A, T383A, T382/3A, T382E, T383E, and T382/3E phosphorylation site mutants, standard PCR-based site-directed mutagenesis was used. The authenticity of all constructs was confirmed by nucleotide sequencing analysis. All PTEN mutant cDNAs were restriction-digested with BamHI and EcoRI enzymes and fused in-frame in the NH2 terminus to an AU5-epitope tag present in the expression vector, pCEFL KZ AU5. The HA-tagged ubiquitin expression plasmid, pCMV-HA-Ub, was a gift from Dr. Zeev Ronai (Mt. Sinai School of Medicine, New York, NY) and was described previously (15) . A Flag-tagged MAGI-2 (also referred to as AIP1 or ARIP1) expression construct (pS{alpha}FlagN-MAGI-2) was generated by fusing a ~3.5 kb EcoRI-EcoRV cDNA fragment to a Flag-epitope containing expression vector, pS{alpha} (16) . For the creation of a GST-fusion protein of the PDZ2 domain of MAGI-2, two restriction enzyme-tagged primers were used to amplify a 285-bp region corresponding to the codon residues from 594 to 688 of MAGI-2. The amplified product was restriction digested with BglII and EcoRI and subcloned into the BamHI and EcoRI sites of the pGEX-KG expression vector.

Antibodies.
The anti-HA (12CA5) monoclonal antibody was derived from the Monoclonal Core Facility of the Mount Sinai School of Medicine. The anti-PTEN mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the anti-actin mouse monoclonal antibody (Santa Cruz Biotechnology), the anti-AU5 monoclonal antibody (Covance, Richmond, CA), the anti-Flag monoclonal antibody (Sigma Chemical Co., St. Louis, MO), the anti-phos-threonine rabbit polyclonal antibody (Sigma Chemical Co. and Zymed Labs, Inc., South San Francisco, CA) were purchased from commercial sources. The anti-PTEN rabbit polyclonal antibody was generated by immunizing rabbit with a fusion protein of GST and the COOH-terminal 216 amino acids of PTEN.

PTEN-MAGI-2 Binding Assay.
For testing binding between MAGI-2 and PTEN mutants, 5 µg of pS{alpha}FlagN-MAGI-2 was transiently transfected into 5 x 106 293T cells per 100-mm culture dish. Approximately 36 h after transfection, cells were solubilized with 700 µl of standard radioimmunoprecipitation assay buffer, and Flag-tagged MAGI-2 was affinity purified using 20 µg of anti-Flag antibody coupled to 150 µl of {gamma}-bind G-Sepharose beads (Pharmacia, Piscataway, NJ). To generate PTEN substrates for the binding experiments, 2 x 106 293T cells in a 100-mm dish were transfected with 5–10 µg of various PTEN expression constructs. Approximately 24 h after transfection, cells were solubilized in 700 µl of a lysis buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA (pH 8.0), 1 mM Na3VO4, 1 mM Na3P3O4, 10 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, and 1 mM aprotinin. For a typical binding assay, 30 µl of the Flag-MAGI-2-containing beads was incubated with ~1 mg of total cell extract expressing similar levels of individual PTEN mutants. Binding was carried out by turning the reactions at 4°C for 2 h and then washing three times with lysis buffer. Bound PTEN proteins were eluted by boiling in 60 µl of a Laemmli sample buffer. An equivalent amount of each binding reaction was loaded on a 12.5% SDS-PAGE gel with subsequent standard Western blot analysis using a rabbit anti-PTEN polyclonal antibody. To test the ability of the PDZ2 domain of MAGI-2 to bind to different PTEN mutants, the fusion protein GST-MAGI2-PDZ2 was synthesized and affinity purified as described previously (17) . For a typical binding experiment, we used 25 µl of glutathione beads coupled with ~30 µg of GST-MAGI2-PDZ2 fusion protein. Binding reactions with various PTEN mutants and subsequent washing conditions were essentially the same as described above.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussions
 REFERENCES
 
Identification of Two Potential Phosphorylation Sites in PTEN Protein at Codons 382 and 383.
We have demonstrated previously that the PTEN COOH terminus plays a role in conferring protein stability (14) . As has been well-documented in the case of p53 tumor suppressor protein, phosphorylation plays a critical role in the regulation of proteosome-mediated protein degradation (18) . We speculated that PTEN may also use a similar mechanism, and an attempt was made to identify potential phosphorylation sites within the COOH-terminal 33 amino acids of PTEN. Using a panel of commercially derived phospho-specific antibodies, the phosphorylation states of PTEN protein overexpressed in cultured cells were investigated. We failed to detect both serine and tyrosine phosphorylation using these immunological reagents (data not shown). However, a positive signal was registered when an {alpha}-p-Thr was used (Fig. 1)Citation . Similar results were also obtained when a different commercially available antibody was tested under similar experimental conditions. However, this p-Thr signal was not detected using bacterially generated PTEN proteins, suggesting that the immunoreactivity observed was most likely attributable to bona fide phosphorylation events in cultured cells (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1. PTEN is phosphorylated on threonine residues. Different AU5-epitope-tagged PTEN constructs representing the WT (WT), phosphatase-defective (AA), COOH-terminal deletion mutants lacking 4 (C{Delta}4) or 33 (C{Delta}33) amino acids, and three phosphorylation site mutants (T382A, T383A, and T382/3A) were ectopically expressed in 293T cells. PTEN proteins were immunoprecipitated from total cell extracts with an anti-AU5 monoclonal antibody and resolved on an SDS 12.5% polyacrylamide gel. Western blot analysis was carried out using an {alpha}-p-Thr (top) or an anti-PTEN rabbit polyclonal antibody (bottom).

 
To identify the threonine residues in question, we performed similar transient transfection experiments using various COOH-terminal truncated PTEN mutants as described previously (14) . As shown in Fig. 1Citation , deleting the 4-amino acid PDZ-binding domain from the PTEN COOH-terminus (C{Delta}4) did not significantly affect the magnitude of {alpha}-p-Thr reactivity, irrespective of whether PTEN was in its WT or AA states. However, deleting 33 amino acids of PTEN COOH-terminus (C{Delta}33) abrogated all of the p-Thr signal. The fact that there were only two threonine residues within this region at codon 382 and 383 (Thr-382 and Thr-383) led us to believe that they may serve as targets for phosphorylation. To test this possibility, we rendered these two threonine residues either singly (T382A and T383A) or doubly (T382/3A) defective for phosphorylation by substituting them with alanine residues. Indeed, {alpha}-p-Thr failed to detect a significant signal in all three PTEN mutants (Fig. 1)Citation . The fact that mutating just one of the two threonines resulted in a nearly complete abrogation of the cross-reactivity suggests that {alpha}-p-Thr showed a higher affinity toward the doubly phosphorylated PTEN molecules. Alternatively, phosphorylation at these threonine residues may show a certain mutual codependency.

PTEN Phosphorylation Site Mutants Affect PTEN Protein Stability.
To address whether phosphorylation events at Thr-382 and Thr-383 could alter PTEN protein stability, transient transfection experiments were performed in a PTEN-null prostate cancer cell line, PC3. As shown in Fig. 2ACitation , when an equal amount of expression vector was transfected, there was a ~10–20 fold reduction in the levels of T382A and T383A mutants when compared with the wild-type protein 24 h after transfection. The double phosphorylation-defective mutant, T382/3A, also was less expressed but consistently displayed a higher expression level than the single-site mutants. All three phosphorylation-defective mutants were almost undetectable 48 h after transfection. In contrast, the three phosphomimic mutants (T382E, T383E, and T382/3E) still retained considerable levels of protein expression at the 48 h time point.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Non-phosphorylated forms of PTEN are unstable. A, equal amounts of indicated plasmids were transfected into a PTEN-null PC3 cell line. Cultures were solubilized at 24 or 48 h, and equal amount of cell extracts were resolved on a SDS 12.5% polyacrylamide gel. B, similar transfection reactions were performed, and 24 h after transfection, cultures were exposed to ALLN for 0, 2, and 4 h before solubilization. The expression levels of various PTEN isoforms were monitored with an anti-PTEN antibody, and protein loading was normalized with an anti-actin antibody.

 
To explore whether the observed decreases in the expression levels of PTEN phosphorylation site mutants were attributable to a proteosome-mediated protein degradation pathway, we performed similar transient transfection expression analysis in the presence or absence of the proteosome inhibitor, ALLN. Transfected cells were treated with either control solvent, DMSO, or 40 µM of ALLN for 2 and 4 h. Whereas the levels of PTEN-WT were not significantly affected by ALLN, all three phosphorylation site mutants displayed a 2–5 fold increases in protein expression with time (Fig. 2B)Citation .

One of the critical steps in the proteosome degradation pathway is the formation of a ubiquitin-protein conjugate (19) . The covalent addition of multiple ubiquitin molecules to the target protein is prerequisite for its degradation by the 26S proteosome. To test whether PTEN was ubiquitinated, we cotransfected various forms of PTEN with a HA-Ub expression construct in 293T cells. Transfected cultures were either treated with or without ALLN for 4 h before cell lysis. Next, PTEN proteins were immunoprecipitated from total cell extracts, and covalently linked HA-Ub was detected by immunoblot with an anti-HA antibody. As expected, the characteristic multimeric banding patterns of ubiquitinated-protein conjugates were clearly demonstrated (Fig. 3)Citation . The observed result was specific, because a similar HA-Ub ladder was not detected in cells transfected with a vector control (data not shown). In addition, the more rapidly degraded T382A and T383A mutants displayed an apparently greater intensity of the HA-Ub conjugation events than either the WT or the phosphomimic mutant T382/3E. Furthermore, in all cases, the addition of ALLN augmented the magnitude of polyubiquitination significantly. On the basis of all these results, we conclude that dephosphorylation at Thr-382 and Thr-383 leads to the increases in PTEN degradation potentially mediated by the ubiquitin-proteosome pathway.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 3. PTEN is polyubiquitinated in vivo. Transient transfection was carried out in 293T cells by coexpressing the indicated PTEN expression plasmids with (+) or without (-) an HA-tagged ubiquitin (HA-Ub) construct. Approximately 48 h after transfection, cultures were treated with (+) or without (-) ALLN for 4 h before solubilization. PTEN proteins were immunoprecipitated (Ip) from total cell extracts with an anti-AU5 monoclonal antibody and Western blot (WB) analysis was carried out to detect the polyubiquitinated chains using an anti-HA antibody (top). The expression levels of PTEN were monitored with an anti-PTEN rabbit polyclonal antibody (bottom). *, position of PTEN. The cross-reactive mouse immunoglobulin heavy chain is indicated.

 
PTEN Phosphorylation Site Mutants Bind MAGI-2 PDZ2 Domain with Elevated Affinity.
Previous experiments have suggested that, by recruiting PTEN to the cell-cell junction, the MAGUK family of proteins, such as MAGI-2, enhances PTEN tumor-suppressor function (12) . The proximity of Thr-382 and -383 to the PDZ-binding domain led us to test whether PTEN binding to MAGI-2 could be regulated by phosphorylation of these residues. For this, we separately overexpressed a Flag-tagged MAGI-2 and various PTEN mutants in 293T cells. Binding reactions were carried out by adding cell extracts expressing similar amounts of various PTEN mutants to an affinity column with bound Flag-MAGI-2. Interestingly, all three phosphorylation-defective mutants displayed a strikingly greater binding capacity of 15–30-fold over their WT counterpart (Fig. 4A)Citation . As expected, the PTEN mutant lacking the 4-amino acid PDZ-binding domain (PTEN-C{Delta}4) failed to interact with Flag-MAGI-2.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 4. Non-phosphorylated forms of PTEN bind MAGI-2 with higher affinity. A, Flag epitope-tagged MAGI-2 and indicated PTEN expression plasmids were separately expressed in 293T cells. Binding reactions were carried out as described in "Materials and Methods." The levels of PTEN bound to Flag-MAGI-2 (Binding) and the relative levels of PTEN added to the binding reactions (Input) were monitored using an anti-PTEN antibody. The amount of Flag-MAGI-2 immobilized onto the affinity columns was detected using an anti-Flag monoclonal antibody (bottom). B, similar binding assays were performed, but GST-MAGI2-PDZ2 fusion proteins immobilized on glutathione beads were used instead. Western blot (WB) analysis was carried out as described in A, and the amount of GST-MAGI2-PDZ2 fusion proteins was visualized by Coomassie Blue stain (bottom). The extent of binding was quantified by an imaging densitometer, and data (top) are expressed as relative binding affinity. Bars, SDs derived from a representative experiment performed in triplicate, which has been repeated at least two times with similar results obtained.

 
Because it has been reported that PTEN binds with the highest affinity to the second PDZ domain of MAGI-2 (MAGI2-PDZ2; Ref. 12 ), we sought to test whether different PTEN phosphorylation site mutants also exhibited differential binding to this domain. For this, we developed an in vitro binding assay using a recombinant GST-MAGI2-PDZ2 fusion protein immobilized on glutathione beads. We performed extensive control experiments, showing that PTEN expressed in cells did not bind either to control glutathione beads or to GST alone (data not shown). Also, as would have been expected, GST-MAGI2-PDZ2 failed to bind to the PTEN-{Delta}4 mutant. More importantly, we were able to affinity purify endogenous PTEN from total cell extracts derived from NIH3T3 fibroblasts, attesting to its suitability as an affinity probe.4

Next, we performed similar binding assays using a panel of PTEN phosphorylation site mutants. We observed that the three phosphorylation-defective mutants of PTEN displayed a 2–5-fold greater relative binding to GST-MAGI2-PDZ2 than the WT protein (Fig. 4B)Citation . Furthermore, the double mutant, T382/3A, showed a consistently higher binding capacity in multiple experiments. In contrast, all of the three phosphomimic mutants (T382E, T383E, and T382/3E) bound to GST-MAGI2-PDZ2 at a level similar to that of the PTEN-WT protein. We conclude from these experiments that dephosphorylation of PTEN at Thr-382 and -383 increases interaction with MAGI-2 most likely through the PDZ2 domain. Also, it is possible that the recruitment of PTEN to the cell-cell junction by MAGI-2 is promoted by dephosphorylation events.


   Discussions
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussions
REFERENCES
 
In this study, we report the identification of two threonine phosphorylation sites in the PTEN COOH terminus that regulate both protein stability and its interaction with MAGI-2. Our data are very similar to two recent reports by Vazquez et. al. (20) and Torres et. al. (21) , in which both groups have identified the same two residues as being phosphorylated in vivo. Furthermore, these two studies also reported additional phosphorylation sites at Ser-370, Ser-380, and Ser-385. Our inability to detect these serine phosphorylation sites could be explained by the limitation of the commercially available anti-phosphoserine antibodies. However, analysis of the individual phosphoacceptor site mutants indicates that only Ser-380, Thr-382, and Thr-383 are more critical in controlling PTEN protein stability and biological activities (20 , 21) .

It is not known, however, the relative phosphorylation states at individual Ser/Thr residues in PTEN-WT protein under different physiological conditions. Also, we have attempted but failed to demonstrate that the phosphorylation events could be modified under different cell culture conditions. These include mitogen stimulation, adherent verses nonadherent growth, and exposure to different stress conditions. On the basis of comparisons of the relative binding capacity between WT and phosphorylation-site mutants to MAGI-2, we estimate that ~95% of PTEN in a cell undergo phosphorylation at one or both of the residues. Therefore, we speculate that the majority of PTEN in vivo are phosphorylated, cytoplasmically localized, relatively stable, but functionally inert. Intriguingly, we consistently observe the double mutant (T382/3A) to be more stable than the two single phosphorylation site mutants (T382A and T383A; Fig. 2Citation ) in our experiments. Coincidentally, the double mutant also displays a greater binding affinity toward MAGI-2 than the single-site mutants (Fig. 4)Citation . Therefore, it is tempting to speculate that PTEN binding to MAGI-2 may have some stabilization effect that could be crucial for its phosphatase function at the cell-cell junction.

Data generated in the present study as well as others (20 , 21) have indicated strongly that phosphorylation of the COOH-terminal PEST domain of PTEN alters its protein stability. Furthermore, we have extended these findings by providing the first evidence that PTEN may undergo ubiquitination. It appears paradoxical that proteosome inhibitor treatment did not significantly alter the PTEN-WT protein level. This can be explained by our earlier estimation that over 95% of PTEN in a cell exists in a relatively stable phosphorylated form with a reported half-life of ~4 h (20, 21, 22) . Thus, the treatment with ALLN would not be expected to elevate further the level of total PTEN protein. However, the small pool of hypophosphorylated and unstable PTEN is revealed in the form of ubiquitinated species when PTEN-WT is overexpressed in 293T cells (Fig. 3)Citation . Whether PTEN undergoes ubiquitination under physiological conditions is unclear at present.

It is predicted that the COOH-terminal 50-amino acid region of PTEN, where the Ser/Thr sites reside, is relatively unstructured (10) . The flexible nature of this motif may well play a crucial role in the regulation of PTEN tumor suppressor functions through phosphorylation. For example, a hypophosphorylated, less-structured PTEN COOH terminus may relieve some of the steric hindrance that would normally prevent the substrate PIP3 from occupying the catalytic pocket. Alternatively, a flexible PTEN COOH-terminus may render the PDZ-binding domain more exposed, making it available for MAGI-2 binding. Our data tend to support the latter hypothesis, because the T382A/T383A mutants display a 15–30-fold greater binding capacity to MAGI-2 than their WT counterpart. Our findings, therefore, could partly explain why PTEN phosphorylation site mutants described by Vazquez et. al. (20) are relatively more active than WT protein in suppressing FKHR transcriptional activity and inducing G1 arrest (20) .

On the basis of the results derived from the present study as well as other published data, it is possible then to postulate an activation model for PTEN. In response to an as yet unknown stimulus, PTEN undergoes dephosphorylation of its COOH-terminal tail and is recruited to the cell-cell junction by MAGI-2. Membrane-localized PTEN then mediates the dephosphorylation of its target substrate, PIP3. After releasing from the MAGI-2 complex, PTEN may either be degraded rapidly through a proteosome-dependent pathway or undergo phosphorylation and revert back to the pool of stable PTEN in the cytoplasm.

Obviously, there are still several outstanding questions as to how PTEN phosphorylation is regulated. For example, the physiological signals that are responsible for activating PTEN have yet to be identified. It still remains to be determined whether PTEN can undergo self-dephosphorylation. Also, are there other PTEN kinases in addition to the reported protein kinase CK2 (21) ? Finally, it is crucial to understand whether the interaction between PTEN and MAGI-2 is necessary for tumor suppression. In fact, whether MAGI-2 possesses tumor suppressor function has not yet been determined. Developing effective means to disrupt MAGI-2-PTEN interaction will certainly address some of these exciting questions.


   FOOTNOTES
 
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.

1 Funded by National Cancer Institute Training Grant CA78207 (to A. C. K.). This work was also partly supported by NIH Grant CA78509. A. M. L. C. is a recipient of a Career Scientist Award from the Irma T. Hirschl Foundation. Back

2 To whom requests for reprints should be addressed, at Cancer Center, Mount Sinai School of Medicine, 1425 Madison Avenue, New York NY 10029. Phone: (212) 659-5490; Fax: (212) 849-2446; E-mail: andrew.chan{at}mssm.edu Back

3 The abbreviations used are: PI, phosphatidylinositide; PI3-K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5 triphosphate; MAGUK, membrane-associated guanylate kinase; WT, wild type; AA, catalytic-inactive (phosphatase-defective); GST, glutathione S-transferase; {alpha}-p-Thr, anti-phospho-threonine antibody; HA-Ub, hemagglutinin-tagged ubiquitin. Back

4 Unpublished observations. Back

Received 3/19/01. Accepted 5/14/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussions
 REFERENCES
 

  1. Li J., Yen C., Liaw D., Podsypanina K., Bose S., Wang S. I., Puc J., Miliaresis C., Rodgers L., McCombie R., Bigner S. H., Giovanella B. C., Ittmann M., Tycko B., Hibshoosh H., Wigler M. H., Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (Wash. DC), 275: 1943-1947, 1997.[Abstract/Free Full Text]
  2. Li D. M., Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor ß. Cancer Res., 57: 2124-2129, 1997.[Abstract/Free Full Text]
  3. Steck P. A., Pershouse M. A., Jasser S. A., Yung W. K., Lin H., Ligon A. H., Langford L. A., Baumgard M. L., Hattier T., Davis T., Frye C., Hu R., Swedlund B., Teng D. H., Tavtigian S. V Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet., 15: 356-362, 1997.[Medline]
  4. Parsons R. Phosphatases and tumorigenesis. Curr. Opin. Oncol., 10: 88-91, 1998.[Medline]
  5. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature (Lond.), 387: 673-676, 1997.[Medline]
  6. Di Cristofano A., Pandolfi P. P. The multiple roles of PTEN in tumor suppression. Cell, 100: 387-390, 2000.[Medline]
  7. Maehama T., Dixon J. E. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem., 273: 13375-13378, 1998.[Abstract/Free Full Text]
  8. Stambolic V., Suzuki A., de la Pompa J. L., Brothers G. M., Mirtsos C., Sasaki T., Ruland J., Penninger J. M., Siderovski D. P., Mak T. W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell, 95: 29-39, 1998.[Medline]
  9. Myers M. P., Pass I., Batty I. H., Van der Kaay J., Stolarov J. P., Hemmings B. A., Wigler M. H., Downes C. P., Tonks N. K. The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA, 95: 13513-13518, 1998.[Abstract/Free Full Text]
  10. Lee J. O., Yang H., Georgescu M. M., Di Cristofano A., Maehama T., Shi Y., Dixon J. E., Pandolfi P., Pavletich N. P. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell, 99: 323-334, 1999.[Medline]
  11. Adey N. B., Huang L., Ormonde P. A., Baumgard M. L., Pero R., Byreddy D. V., Tavtigian S. V., Bartel P. L. Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding. Cancer Res., 60: 35-37, 2000.[Abstract/Free Full Text]
  12. Wu X., Hepner K., Castelino-Prabhu S., Do D., Kaye M. B., Yuan X. J., Wood J., Ross C., Sawyers C. L., Whang Y. E. Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc. Natl. Acad. Sci. USA, 97: 4233-4238, 2000.[Abstract/Free Full Text]
  13. Wu Y., Dowbenko D., Spencer S., Laura R., Lee J., Gu Q., Lasky L. A. Interaction of the tumor suppresser PTEN/MMAC with a PDZ domain of MAGI 3, a novel membrane-associated guanylate kinase. J. Biol. Chem., 275: 21477-21485, 2000.[Abstract/Free Full Text]
  14. Tolkacheva T., Chan A. M. Inhibition of H-Ras transformation by the PTEN/MMAC1/TEP1 tumor suppressor gene. Oncogene, 19: 680-689, 2000.[Medline]
  15. Musti A. M., Treier M., Bohmann D. Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science (Wash. DC), 275: 400-402, 1997.[Abstract/Free Full Text]
  16. Shoji H., Tsuchida K., Kishi H., Yamakawa N., Matsuzaki T., Liu Z., Nakamura T., Sugino H. Identification and characterization of a PDZ protein that interacts with activin type II receptors. J. Biol. Chem., 275: 5485-5492, 2000.[Abstract/Free Full Text]
  17. Osada M., Tolkacheva T., Li W., Chan T. O., Tsichlis P. N., Saez R., Kimmelman A. C., Chan A. M. Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival. Mol. Cell. Biol., 19: 6333-6344, 1999.[Abstract/Free Full Text]
  18. Ashcroft M., Vousden K. H. Regulation of p53 stability. Oncogene, 53: 7637-7643, 1999.
  19. Hershko A., Ciechanover A. The ubiquitin system. Annu. Rev. Biochem., 67: 425-479, 1998.[Medline]
  20. Vazquez F., Ramaswamy S., Nakamura N., Sellers W. R. Phosphorylation of the PTEN tail regulates protein stability and function. Mol. Cell. Biol., 20: 5010-5018, 2000.[Abstract/Free Full Text]
  21. Torres J., Pulido R. The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteosome-mediated degradation. J. Biol. Chem., 276: 993-998, 2001.[Abstract/Free Full Text]
  22. Georgescu M. M., Kirsch K. H., Akagi T., Shishido T., Hanafusa H. The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region. Proc. Natl. Acad. Sci. USA, 96: 10182-10187, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Yang, S. Wang, B. Sung, G. Lim, and J. Mao
Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation
J. Biol. Chem., August 1, 2008; 283(31): 21703 - 21713.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Fouladkou, T. Landry, H. Kawabe, A. Neeb, C. Lu, N. Brose, V. Stambolic, and D. Rotin
The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization
PNAS, June 24, 2008; 105(25): 8585 - 8590.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Selvendiran, L. Tong, S. Vishwanath, A. Bratasz, N. J. Trigg, V. K. Kutala, K. Hideg, and P. Kuppusamy
EF24 Induces G2/M Arrest and Apoptosis in Cisplatin-resistant Human Ovarian Cancer Cells by Increasing PTEN Expression
J. Biol. Chem., September 28, 2007; 282(39): 28609 - 28618.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Odriozola, G. Singh, T. Hoang, and A. M. Chan
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain
J. Biol. Chem., August 10, 2007; 282(32): 23306 - 23315.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
A. Gil, A. Andres-Pons, E. Fernandez, M. Valiente, J. Torres, J. Cervera, and R. Pulido
Nuclear Localization of PTEN by a Ran-dependent Mechanism Enhances Apoptosis: Involvement of an N-Terminal Nuclear Localization Domain and Multiple Nuclear Exclusion Motifs
Mol. Biol. Cell, September 1, 2006; 17(9): 4002 - 4013.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
Y.-G. Chen, Q. Wang, S.-L. Lin, C. D. Chang, J. Chung, and S.-Y. Ying
Activin Signaling and Its Role in Regulation of Cell Proliferation, Apoptosis, and Carcinogenesis.
Experimental Biology and Medicine, May 1, 2006; 231(5): 534 - 544.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. M. Al-Khouri, Y. Ma, S. H. Togo, S. Williams, and T. Mustelin
Cooperative Phosphorylation of the Tumor Suppressor Phosphatase and Tensin Homologue (PTEN) by Casein Kinases and Glycogen Synthase Kinase 3{beta}
J. Biol. Chem., October 21, 2005; 280(42): 35195 - 35202.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Valiente, A. Andres-Pons, B. Gomar, J. Torres, A. Gil, C. Tapparel, S. E. Antonarakis, and R. Pulido
Binding of PTEN to Specific PDZ Domains Contributes to PTEN Protein Stability and Phosphorylation by Microtubule-associated Serine/Threonine Kinases
J. Biol. Chem., August 12, 2005; 280(32): 28936 - 28943.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. M. Connor, S. Subbaram, K. J. Regan, K. K. Nelson, J. E. Mazurkiewicz, P. J. Bartholomew, A. E. Aplin, Y.-T. Tai, J. Aguirre-Ghiso, S. C. Flores, et al.
Mitochondrial H2O2 Regulates the Angiogenic Phenotype via PTEN Oxidation
J. Biol. Chem., April 29, 2005; 280(17): 16916 - 16924.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Okahara, H. Ikawa, Y. Kanaho, and T. Maehama
Regulation of PTEN Phosphorylation and Stability by a Tumor Suppressor Candidate Protein
J. Biol. Chem., October 29, 2004; 279(44): 45300 - 45303.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
D. D. Mruk and C. Y. Cheng
Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis
Endocr. Rev., October 1, 2004; 25(5): 747 - 806.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
A. Merlo and B. Bettler
Glioblastomas on the Move
Sci. Signal., April 20, 2004; 2004(229): pe18 - pe18.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Lu, Q. Yu, J. H. Liu, J. Zhang, H. Wang, D. Koul, J. S. McMurray, X. Fang, W.K. A. Yung, K. A. Siminovitch, et al.
Src Family Protein-tyrosine Kinases Alter the Function of PTEN to Regulate Phosphatidylinositol 3-Kinase/AKT Cascades
J. Biol. Chem., October 10, 2003; 278(41): 40057 - 40066.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Torres, J. Rodriguez, M. P. Myers, M. Valiente, J. D. Graves, N. K. Tonks, and R. Pulido
Phosphorylation-regulated Cleavage of the Tumor Suppressor PTEN by Caspase-3: IMPLICATIONS FOR THE CONTROL OF PROTEIN STABILITY AND PTEN-PROTEIN INTERACTIONS
J. Biol. Chem., August 15, 2003; 278(33): 30652 - 30660.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Wu, X. Wang, W. Zhang, W. Reed, J. M. Samet, Y. E. Whang, and A. J. Ghio
Zinc-induced PTEN Protein Degradation through the Proteasome Pathway in Human Airway Epithelial Cells
J. Biol. Chem., July 18, 2003; 278(30): 28258 - 28263.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
K. A. Waite and C. Eng
BMP2 exposure results in decreased PTEN protein degradation and increased PTEN levels
Hum. Mol. Genet., March 15, 2003; 12(6): 679 - 684.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Vazquez, S. R. Grossman, Y. Takahashi, M. V. Rokas, N. Nakamura, and W. R. Sellers
Phosphorylation of the PTEN Tail Acts as an Inhibitory Switch by Preventing Its Recruitment into a Protein Complex
J. Biol. Chem., December 21, 2001; 276(52): 48627 - 48630.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tolkacheva, T.
Right arrow Articles by Chan, A. M-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tolkacheva, T.
Right arrow Articles by Chan, A. M-L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online