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. 2006 Mar 7;103(10):3633-8.
doi: 10.1073/pnas.0510570103. Epub 2006 Feb 28.

Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane

Francisca Vazquez  1 , Satomi Matsuoka, William R Sellers, Toshio Yanagida, Masahiro Ueda, Peter N Devreotes
Affiliations

Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane

Francisca Vazquez et al. Proc Natl Acad Sci U S A. .

Abstract

The tumor suppressor function of PTEN is strongly linked to its ability to dephosphorylate phosphatidylinositol-3,4,5 trisphosphate and, thereby, control cell growth, survival, and migration. However, the mechanism of action of PTEN in living cells is largely unexplored. Here we use single-molecule TIRF microscopy in living cells to reveal that the enzyme binds to the membrane for a few hundred milliseconds, sufficient to degrade several phosphatidylinositol-3,4,5 trisphosphate molecules. Deletion of an N-terminal lipid-binding motif completely abrogates membrane interaction and in vivo function. Several mechanisms, including C-terminal tail phosphorylations, appear to hold PTEN in a constrained conformation that limits its rate of association with the membrane. The steady-state level of bound PTEN is highest at sites of retracting membrane, including the rear of highly polarized cells. The dynamic membrane association could be modulated temporally or spatially to alter PTEN activity in specific physiological situations and could have important implications for tumor suppressor function.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Confocal microscopy shows cytoplasmic and nuclear localization of human PTEN in HEK293 and D. discoideum cells. (A) HEK293 cells were transiently transfected with the indicated YFP fusion proteins and imaged 48 h later with a confocal laser scanning microscope. (B) D. discoideum cells stably expressing the fusion proteins indicated in the figure were imaged with a confocal microscope. Cells were either under full nutrient conditions (Left) or starved for 5 h to induced them to polarize (Right).
Fig. 2.
Fig. 2.
Single-molecule TIRFM imaging reveals membrane localization of PTEN in Dictyostelium and HEK293 cells. (A) Consecutive frames of cells expressing PTEN-YFP or DdPTEN-YFP observed by TIRFM at 33 ms per frame (see Movies 1–3). Areas outlined within green boxes and magnified insets show appearance and disappearance of individual spots. (B) Quantification of the number of membrane-bound PTEN molecules relative to the average cytosolic fluorescence in D. discoideum cells expressing PTEN, PTENΔPBD-YFP, YFP, or DdPTEN-YFP. The number of spots per μm2 appearing per second detected by TIRFM was divided by the cytosolic fluorescence detected by EPI-FM. Average ± SEM (n ≥ 10). (C) Consecutive frames of cells expressing PTEN or PTENC124S/A4-YFP observed by TIRFM in HEK293 cells (see Movies 4 and 5). Areas outlined within green boxes and magnified insets show appearance and disappearance of individual spots. (D) Quantification of the number of membrane-bound PTEN molecules relative to the average cytosolic fluorescence in HEK293 cells expressing PTEN, PTENΔPBD-YFP, or YFP calculated as in B. Average ± SEM (n ≥ 10).
Fig. 3.
Fig. 3.
Membrane binding is required for PTEN to complement pten− D. discoideum cells. (A and C) D. discoideum pten− cells stably expressing the indicated constructs were plated in nonnutrient agar to induce development. Images were taken 24 h after starvation. (B and D) Expression levels of the specific proteins were determined by Western blot analyses by using anti-GFP antibody for the YFP-tagged constructs or anti-PTEN antibody for the untagged constructs.
Fig. 4.
Fig. 4.
Several mutations alter the steady-state number of PTEN membrane-bound molecules. (A) Relative number of membrane-bound PTEN molecules in D. discoideum cells stably expressing PTEN (WT or mutants). The number of spots detected by TIRFM was divided by the relative cytosolic fluorescence detected by EPI-FM calculated as in Fig. 2B. (B) Subcellular localization of PTEN/WT and mutants in undifferentiated D. discoideum cells observed by confocal microscopy. (C) HEK293 cells transiently transfected with the indicated plasmids and imaged 48 h after with a confocal laser scanning microscope. (D) Confocal images of subcellular localization of PTEN in differentiated polarized cells (Top and Middle) and cells undergoing cytokinesis (Bottom). (E) Trajectories of four molecules of PTEN-YFP tracked at 33 frames per s (see also Table 1).
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