| pmid | 10468583 | |||||
|---|---|---|---|---|---|---|
| title | The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region. | |||||
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| journal | Proc Natl Acad Sci U S A | |||||
| year | 1999 | |||||
| full_text_available | true | |||||
| full_text_extraction_method | html | |||||
| pmcid | PMC17863 | |||||
| doi | 10.1073/pnas.96.18.10182 |
Authors: Georgescu MM, Kirsch KH, Akagi T, Shishido T, Hanafusa H Journal: Proc Natl Acad Sci U S A (1999) DOI: 10.1073/pnas.96.18.10182 PMC: PMC17863
- Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10182-7. doi: 10.1073/pnas.96.18.10182.
The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region.
Georgescu MM(1), Kirsch KH, Akagi T, Shishido T, Hanafusa H.
Author information: (1)Laboratory of Molecular Oncology, The Rockefeller University, New York, NY 10021, USA. georgem@rockvax.rockefeller.edu
PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorage-independent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxyl-terminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxyl-terminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted beta-strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumor-suppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.
DOI: 10.1073/pnas.96.18.10182 PMCID: PMC17863 PMID: 10468583 [Indexed for MEDLINE]
Abstract
PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorage-independent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxyl-terminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxyl-terminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted β-strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumor-suppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.
C-Terminal Mutations Inactivate the Tumor-Suppressor Function of PTEN. In primary tumors and tumor cell lines many PTEN mutations are detected in the phosphatase domain situated in the N terminus of the protein. Little is known about a subset of mutations that occur at a distance from the phosphatase domain in exons 7 and 8. To study the impact of these mutations on PTEN tumor-suppressor function, we introduced in the PTEN cDNA three of the most commonly detected mutations in the exon 8 as well as three sporadically detected mutations in exon 9 (Fig.1). A first mutation introduces a stop codon at position 319 (PTEN-319) and is the most frequent site mutation found in a variety of primary tumors of different origins (17–22). A second mutation is an in-frame 3-nt deletion removing only the codon specifying T319 (PTEN-T319Δ), identified also in some tumors (1,18). A third category of mutations determines frameshifts resulting in stop codons at positions 342 or 343 (1,4,16,17,19,21,23). In this case we introduced directly a stop codon in position 342 (PTEN-342) that eliminates the sequence coded by the last exon of PTEN. The three mutations in exon 9 were L345 to Q (4), T348 to I (21), and a nonsense mutation in position 385 (26). For comparison, we used PTEN-PI, with the inactivating mutation H123-to-Y identified in a tumor (27).
The wild-type and various mutants of PTEN were cloned in the pCX retroviral vector and expressed in the U87 MG glioblastoma cell line that is deficient for PTEN. The anchorage-independent growth of these cells, which reflects their transformed phenotype, was evaluated by the ability to form colonies in soft agar (Fig. 1 A ). Whereas wild-type PTEN efficiently suppressed the formation of colonies of U87 MG cells in soft agar, all the mutants allowed the development of colonies. The size of the colonies varied for the different constructs. Cells expressing the phosphatase-inactive PTEN-PI and the truncated mutants PTEN-319 and PTEN-342 displayed large colonies comparable to those formed by the control U87 MG cells transfected with the vector alone, indicating that these mutants completely abolished the tumor-suppressor function of PTEN. The size of the colonies from cells expressing the point mutants PTEN-T319Δ and PTEN-L345Q was intermediate, and that of PTEN-T348I or the truncated variant PTEN-385 was even smaller than that of control cells, suggesting that these mutations conferred only a partial inactivation of the tumor-suppressor function of PTEN. The size of the colonies generally was correlated to the proliferation rate of cells expressing the various mutants (Fig. 1 B ), indicating that the tumor-suppressor effect of PTEN is due mainly to the arrest of proliferation.
PTEN Contains Two PEST Sequences and a PDZ Motif in the C Terminus. To investigate the mechanism of degradation of PTEN, we searched the coding sequence of PTEN for degradation motifs and detected two putative PEST sequences situated between amino acids 350–375 and 379–396 of the C terminus. The probability that these represent true PEST sequences was very high because their scores were +20.49 and +19.41, which ranked them well above PEST sequences from other proteins, e.g., Fos PEST sequence has a score of +10.1 (28). The role of PEST sequences would be to target proteins with short intracellular half-life for proteolytic degradation, and their deletion usually determines an increased expression level of the protein. In PTEN, deletions affecting both or only one of the PEST sequences in the constructs PTEN-351, PTEN-363, PTEN-379, and PTEN-385 resulted in lower expression levels of the proteins (Fig.3C). The last 3 aa of PTEN, T-K-V, form the consensus (T-X-V-COOH) for binding PDZ domains. Because in the previous constructs the terminal PDZ-binding motif also was deleted, two mutants removing only this motif, PTEN-401 and PTEN-398, also were expressed (Fig.3C). For these, the protein level was similar to the full-length protein, indicating that this motif did not affect the expression level of PTEN. Taken together, these results showed that truncations of the PEST sequences decreased the expression of PTEN, most likely by impairing the folding of the protein.
To assess the importance of these motifs for the tumor-suppressor function of PTEN, the mutants lacking both PEST sequences or the terminal PDZ-binding motif (PTEN-351 and PTEN-401, respectively) were expressed in U87-MG cells and were found to induce effects comparable to wild-type PTEN on cell growth (Fig. 1 ). These data showed that the PEST and PDZ-binding motifs are not essential for the tumor-suppressor function.
The C-Terminal Point Mutations Destabilize the Predicted Secondary Structure. The decreased expression level of the C-terminal mutants could have resulted from misfolding of the protein. To test this hypothesis we searched and found sequences in which elements of secondary structure were predicted with high probability by all the programs used (Fig.4). Most interestingly, these sequences corresponded to the regions where most of the natural mutations were detected. In particular, the point deletion of T319 in exon 8 and the missense mutations in exon 9, L345 to Q and T348 to I, mapped to regions predicted to form β-strands. By running the prediction programs with these mutations in the sequence, the probability value of the β-strand formation was modified (Fig.4), indicating that these mutations destabilized the predicted secondary structure of the protein.
The degree of destabilization of the β-strands rather than the expression level of the mutants correlated to the cell growth phenotype (shown in Fig. 1 ). The T348I mutation that preserves a modified β-strand still suppressed cell growth, although not as efficiently as wild-type PTEN. The mutations T319Δ and L345Q, which have the most destabilizing effect on the β-strands, reversed efficiently the tumor-suppressor phenotype of PTEN.
DISCUSSION
The tumor-suppressor function of PTEN has been associated with its ability to dephosphorylate phosphoinositides at position D3 of the inositol ring and to antagonize the PI 3-kinase-PKB/Akt antiapoptotic pathway ( 10 , 11 ). Mutations occurring in tumors map frequently to the phosphatase region of PTEN situated in the N-terminal part of the molecule. We show here that the C-terminal region of PTEN is also essential for the tumor-suppressor function of PTEN. The mutations detected in tumors in this region cluster mainly in exons 7 and 8, and they result usually in premature termination of the ORF. In some reports, these mutations constitute up to 50% of the mutations detected in tumor specimens ( 4 , 19 , 23 ). By expressing PTEN variants with the most frequently reported mutations in exons 8 and 9 in U87-MG glioblastoma cells, we observed that the tumor growth and proliferation of these cells is no longer suppressed. Two of these variants are truncated proteins at amino acids 319 and 342, and, in both cases, the tumor-suppressing ability of PTEN is completely abolished (see Fig. 1 ). Predictably, the upstream truncations occurring in exon 7 would lead to a similar phenotype.
In a first step to identify the mechanism responsible for the inactivation of the tumor-suppressor function, we observed that, relative to wild-type PTEN, all the C-terminal mutants had decreased protein expression levels because of a more rapid degradation. Rapid degradation is a regulatory mechanism for the inactivation of the tumor-suppressor function that also was described for p53 (reviewed in ref. 29 ). p53 tumor suppressor is polyubiquitinated and degraded rapidly by the proteasome ( 30 ). For PTEN, we could not detect by immunoprecipitation in transfected COS-7 cells ubiquitinated forms of the wild-type or mutant proteins (not shown), suggesting a ubiquitin-independent degradation. Although PTEN contains two PEST sequences, their role in targeting the protein for degradation is not clear. PTEN has a relatively long half-life (more than 2 h), and the deletion of the PEST sequences actually decreased the expression level of the protein in cells. A possibility could be that this truncation affected the structure or the chemical properties of the molecule, leading to its destabilization. It is remarkable that the majority of protein phosphatases contain PEST sequences ( 31 ). Similar to PTEN, it also is not known whether their role is to target these proteins for degradation because the half-lives of some investigated phosphatases are also relatively long ( 32 , 33 ).
Another motif present in the C-terminal region of PTEN is a PDZ-binding motif. Although this motif may have a role in the regulation of PTEN, its deletion did not have a detectable influence on the tumor-suppressor activity of PTEN. It is possible that PTEN is recruited by other mechanisms to the cell membrane, but an alternative explanation could be that the protein lacking the PDZ motif reaches the membrane because of overexpression in the stably transfected U87-MG cells.
The deletion of both PEST and PDZ-binding sequences did not have a significant effect on the tumor-suppressor function of PTEN, but a truncation of only nine residues upstream completely inactivated it (see mutants PTEN-351and PTEN-342 in Fig. 1 ). We also observed that most of the mutations arising in tumors map within a 50-aa region immediately upstream the PEST sequences. The mechanism responsible for the inactivation of the tumor-suppressor function of PTEN became clear when variants with minor sequence modifications were investigated. Two point mutants (positions 345 and 348) and a single residue deletion mutant (position 319) displayed reduced expression levels in cells and reversed to different extents the tumor-suppressor activity of PTEN. Amino acid 319 is in the +4 position from a tyrosine that could represent a putative phosphorylation site ( 1 ). By using antiphosphotyrosine antibodies, we could not detect tyrosine phosphorylation of PTEN or PTEN-PI in cells stimulated with epidermal growth factor (not shown), suggesting that the phenotypic change does not relate to changes in tyrosine phosphorylation. Because proteins with defects in folding become unstable ( 34 ), we suspected that these three mutations interfere with the structure of PTEN. We found that all 3 aa are located in two predicted β-strands. Moreover, the growth phenotype of the mutants correlates with the degree of disruption of these β-strands. The presence of the last β-strand in the variant deleted at position 351 (PTEN-351) and its elimination in the shorter variant, PTEN-342, would explain the difference in phenotype between them. The actual configuration of predicted secondary structure elements in the context of the whole molecule also depends on the interactions with other structural elements ( 35 ). To determine the exact folding of these amino acid stretches in their environment and the conformational changes induced by the mutations detected in tumors, it is necessary to perform three-dimensional analysis. By such analysis, mutations from tumors affecting the folding of another tumor-suppressor protein, p16(INK4) ( 36 ), were mapped to regions conferring stability to the protein ( 37 ).
Although the destabilization of the C-terminal structure could explain the decreased expression level, it could not explain why mutants with similar expression levels had different effects on the phenotype (compare PTEN-L345Q with PTEN-T348I in Fig. 1 ). The answer came from the examination of the phosphatase activity that was affected differentially by these mutants. These data indicated that mutations in the C-terminal region inactivate the tumor-suppressor function of PTEN by affecting its intrinsic phosphatase activity most likely as a result of conformational changes. This effect is reflected also by the persistence of PKB/Akt activation in cells expressing these mutants. Although the boundaries of the PTEN phosphatase domain have been established by analogy to other protein phosphatases ( 27 ), we show here that the presence of a correct folding in the C terminus is essential for the phosphatase activity. Apparently, the effect of the C-terminal mutations on the phosphatase activity can be modulated in cellular context, as shown for the mutant PTEN-T348I (see Fig. 5 ), suggesting another important regulatory role for the C-terminal region. We are investigating at present which cellular component is necessary for the recovery of the enzymatic activity because this approach could have therapeutic implications for correcting PTEN misfolding.
In conclusion, the C-terminal region of PTEN contains predicted secondary structure elements that are essential for the tumor-suppressor function of the protein. PTEN variants with disruptions of these structures are degraded rapidly, have impaired enzymatic activity, and allow tumor growth of the cells. The effects on the stability and enzymatic function as well as the perfect conservation in mammals of the C-terminal region call for an important complementary study, which is to determine the three-dimensional organization of the whole protein.