Figure 7:

LIMK2 destabilizes PTEN in PC3 cells and promotes metastatic phenotypes. (A) 5A-PTEN is expressed at higher-level compared to WT-PTEN in PTEN-deficient PC3 cells. HA-tagged wild-type PTEN or 5A-PTEN were ectopically expressed in PC3 cells and protein levels of PTEN, LIMK2 and actin were analyzed using their respective antibodies. (B) Histogram shows average values of wild type and mutant PTEN obtained from three independent experiments. Data shown are mean ± SEM. ***P <0.0005 as compared to control cells. (C) LIMK2-mediated phosphorylation of PTEN decreases its stability. PTEN levels were analyzed in PTEN-PC3 and 5A-PTEN-PC3 cells treated with cycloheximide (10 μM) for 10 and 20h. (D) Graphical representation of PTEN half-life in PTEN-PC3 and 5A-PTEN-PC3 cells. **P <0.005. (E) LIMK2 overexpression increased the ubiquitylation of WT-PTEN, but not 5A-PTEN in PC3 cells. (F) Expression of WT and 5A-PTEN impaired cell proliferation in PC3 cells. PC3, LIMK2-PC3, PTEN-PC3 and 5A-PTEN-PC3 cells were cultured in 96-well plates for 24, 48, and 72h and subjected to MTT assay. (G) Data showing increased cell proliferation in PTEN-PC3 and 5A-PTEN-PC3 on LIMK2 overexpression. (H) LIMK2 depletion substantially reduced cell growth in PTEN-PC3 and 5A-PTEN-PC3 cells. (I) PTEN prevents colony formation in a soft agar assay in PC3 cells. *p < 0.05 as compared to vector-expressing control. (J) PTEN inhibits cell motility in PC3 cells. *p < 0.05 as compared to vector control. (K) Representative images of chemotaxis assay, Magnification, 200×. (L and M) LIMK2 overexpression increases cell motility in both PTEN-PC3 and 5A-PTEN-PC3 cells. (N and O) LIMK2 depletion inhibits cell motility in both PTEN-PC3 and phospho-resistant 5A-PTEN-PC3 cells.