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. 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

M M Georgescu  1 , K H Kirsch, T Akagi, T Shishido, H Hanafusa
Affiliations

The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region

M M Georgescu et al. Proc Natl Acad Sci U S A. .

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 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.

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Figures

Figure 1
Figure 1
(A) Failure of PTEN C-terminal mutants to suppress the anchorage-independent growth of U87-MG cells. Retrovirus-infected cells stably expressing PTEN or the indicated mutants (the names of the mutants are abbreviated by the mutation only) were allowed to form colonies in soft agar. The photographs were taken with an Axiovert 135 microscope (Zeiss) at ×10 magnification. (B) The tumor growth of U87-MG cells was assessed as in A, and the size of colonies was scaled from no growth (−) to maximum growth that is similar to vector-transfected cells (+++). The proliferation represents the number of retrovirus-infected U87-MG cells surviving after the completion of the drug selection. These experiments were repeated at least three times with similar results.
Figure 2
Figure 2
Reduced protein stability of PTEN C-terminal mutants. (A) Proteins (50 μg) from lysates of stably transfected U87-MG (U87) cells were resolved by SDS/PAGE and analyzed by immunoblotting with anti-Myc antibodies recognizing Myc-tagged PTEN and mutants. Arrowheads indicate the position of low-molecular-weight variants of PTEN. The point mutants of PTEN have low expression levels and are not evident in total lysates, but they can be demonstrated by immunoprecipitation (not shown). (B) Analysis of extracted mRNAs from the same cells by reverse transcription and PCR with primers specific for Myc-tagged PTEN (Upper) or actin (Lower).
Figure 3
Figure 3
Rapid degradation of PTEN C-terminal mutants (A and B). Pulse–chase assays were performed in COS-7 cells transfected with FLAG-tagged PTEN and mutants. Cells were pulse–radiolabeled for 30 min and chased for the time periods indicated, up to 2 h in A or up to 4 h in B. Proteins were immunoprecipitated from lysates with the M2 antibody, and the filter first was exposed (Upper) and then immunoblotted with the M2 antibody to monitor the amount of immunoprecipitated proteins (Lower). The arrowhead indicates the Ig heavy chain. The densitometric analysis using a Molecular Imager System (Bio-Rad) is shown in the graphs (♦, ■, +, and ▴ represent PTEN, PTEN-T319Δ, PTEN-319, and PTEN-342, respectively). (C) The expression level of the PTEN C-terminal mutants is shown in total lysates (50 μg of protein) from transiently transfected 293T cells analyzed by immunoblotting with the M2 antibody.
Figure 4
Figure 4
The PTEN C-terminal mutants map to predicted β-strands. The predicted secondary structures (SS), β-strand (b and double-headed arrows) and loop (L), are shown for PTEN and the variants, with point deletion (Δ) in position 319 or point mutations in positions 345 and 348. The changed amino acids and β-strands are shown in bold. The numbers indicate the probability, scaled from 0 to 9, for assigning β-strands (pr:b) by the program predictprotein from European Molecular Biology Laboratory, Heidelberg.
Figure 5
Figure 5
The phosphatase activity of the PTEN C-terminal mutants. Shown is a phosphatase assay using 2.5 μg glutathione S-transferase-fusion proteins (A) or immunoprecipitated proteins from lysates of 293T cells transfected with FLAG-tagged PTEN and mutants (B). (B Lower) The amount of the immunoprecipitated proteins that, at the end of the phosphatase reaction, were resolved on SDS/PAGE and analyzed by immunoblotting with M2 antibody. The amount of free phosphate released in the reaction was measured in a colorimetric assay and compared with a standard curve. (C) PKB/Akt phosphorylation in U87-MG cells stably expressing PTEN and the indicated mutants. Proteins (50 μg) from total lysates were analyzed by immunoblotting with anti-PKB/Akt antibodies recognizing total levels of PKB/Akt (Akt) or only the phosphorylated form (P-Akt). These experiments demonstrated repeatedly similar results.
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