Nuclear β-catenin is mainly regulated by PTEN and ILK via GSK-3. The expression of nuclear β-catenin is dramatically and comparably abrogated by expression of PTEN (82%), ILK-KD (79%), and GSK-3-WT (78%) into PC3 cells. Nuclear β-catenin expression is altered to a lesser extent by the dominant negative PKB-AAA (79%) and completely unaffected by expression of ILK-WT (78%).Values in brackets indicate the transfection efficiency of the various plasmids. The absence of any alterations in the total cellular β-catenin expression demonstrates the specific effect of the various components upon nuclear β-catenin.

Schematic representation of the regulation of cyclin D1 from integrins and growth factor receptors via ILK and GSK-3. ILK activity is regulated in a PI-3 kinase–dependent manner and activated ILK regulates β-catenin and subsequently cyclin D1, and therefore cell proliferation, mainly via direct phosphorylation of GSK-3 and to a lesser extent via PKB. Stimulation of ILK results in a phosphorylation-mediated inhibition of GSK-3 activity. Inhibition of GSK-3 can lead to the accumulation of β-catenin, which activates the cyclin D1 gene by complexing with transcription factors of the TCF/LEF-1 family and binding to the TCF/LEF-1 binding site in the cyclin D1 promoter. PTEN suppresses the expression of cyclin D1 by inhibiting PI-3 kinase–dependent activation of ILK.

PTEN stimulates phosphorylation of β-catenin and enhances its rate of degradation. (A) PC3 cells transfected with empty vector (control) or PTEN were starved with DME without cysteine and methionine, pulsed with [³⁵S]promix for 1 h, and then chased for the indicated time-points with cold DME containing cysteine/methionine and FBS. Cells were then harvested, lysed, and immunoprecipitated for β-catenin. Results were analyzed by densitometry and expressed as a percentage of the value at 0 h. (B) In correlation with the enhanced degradation of β-catenin on reexpression of PTEN in PC3 cells, the phosphorylation at ser33/37 and thr41 of β-catenin also increases with transfection of increasing amounts of PTEN into PC3 cells.

Expression of nuclear β-catenin is reduced by reexpression of PTEN in PTEN null prostate cancer cells. (A) Immunofluorescence analysis of PTEN null prostate cancer cells, PC3, shows that a significant population (70%) of these cells express high levels of β-catenin in their nucleus (arrowheads). Reexpression of PTEN in PTEN null prostate cancer cells LNCaP (B) and PC3 (C) results in a dose-dependent reduction in the expression of nuclear β-catenin. (D) Immunofluorescence analysis of β-catenin confirms that unlike nontransfected PC3 cells (arrowheads), PC3 cells transfected with PTEN (arrows) exhibit anuclear localization β-catenin. A significantly lower proportion of cells in this case express high levels of nuclear β-catenin (16%). Bar, 5 μm.

Alteration in the expression level of cyclin D1 and the cyclin-CDK inhibitory proteins p27^Kip and p21^Cip, in serum-starved PC3 cells transiently transfected with empty vector (control), ILK-WT (78%), ILK-KD (79%), PTEN-WT (82%), or GSK-3-WT (78%). Values in brackets indicate the transfection efficiency of each plasmid measured as described in Materials and Methods. Cells transfected with the various plasmids were deprived of serum for 18 h, commencing 48 h posttransfection. Equal amounts of protein from the various treatments were then analyzed for cyclin D1, p27^Kip, and p21^Cip expression by Western blot using the respective antibodies. Although cyclin D1 expression is constitutively elevated in a serum-independent manner in PC3 cells transfected with either empty vector or ILK-WT, it was decreased in cells transfected with ILK-KD, PTEN-WT, or GSK-3-WT. The p27^Kip and p21^Cip content was unaffected by all the plasmid transfections. ILK-WT-V₅, ILK-KD-V₅, PTEN-WT-GFP, and GSK-3-WT-HA transgene expressions were determined in the transfectants by Western blot analysis with anti-V₅, anti-GFP, and anti-HA antibodies, respectively.

(A) Transcription of cyclin D1 is inhibited by PTEN-WT, ILK-KD, and GSK-3. Effect of PTEN, ILK, and GSK-3 on cyclin D1 promoter activity. PC3 cells were cotransfected with pGL3-cyclin D1 and empty vector, ILK-WT, ILK-KD, PTEN-WT, or GSK-3-WT. Cells were serum starved for 18 h commencing 48 h post transfection, refed with serum for 1 h, lysed, and assessed for cyclin D1 activity by a luciferase reporter gene assay. Bar graph demonstrates that cyclin D1 promoter activity is significantly reduced by comparable magnitude by the reexpression of PTEN or expression of ILK-KD and GSK-3-WT. Bottom panels show the comparable enhancement of the expression of ILK, PTEN, and GSK-3 upon transfection of ILK-WT or ILK-KD, PTEN-WT, and GSK-3-WT, respectively. (B) Northern blot of cyclin D1 in serum-starved PC3 cells. Total RNA was prepared by the TRIZOL method from serum-starved PC3 cells transfected with empty vector (control), ILK-KD, PTEN-WT, and GSK-3-WT. Transcriptional expression determined by probing the blot with a cyclin D1 probe randomly labeled with [³²P]dCTP demonstrated highly comparable reductions due to transfection of PTEN, ILK-KD, or GSK-3-WT.

PTEN and ILK-KD inhibit DNA binding activities of β-catenin–TCF complex. (A) Electrophoretic mobility shift assays, using an oligonucleotide containing a potential binding site for TCF, demonstrated an abundance of DNA–protein complex in empty vector–transfected PC3 cells (panel 1, lane 1). The quantities of the protein–DNA complex are reduced significantly in PC3 cells transfected with ILK-KD or PTEN (panel 1, lanes 2 and 3). Electrophoretic mobility shift assays performed in the presence of a supershifting antibody (anti–β-catenin antibody) confirmed that the transcription factor complex binding to the oligonucleotide includes β-catenin (panel 2). (B) Coimmunoprecipitation of TCF-4 or Lef-1 with β-catenin using nuclear lysates from empty vector (control), ILK-KD–, or PTEN-WT–transfected PC3 cells show reduced complex formation in ILK-KD– and PTEN-WT–transfected cells compared with control cells. (C) The relative β-catenin/TCF activities (TOPFLASH/FOPFLASH luciferase reporter) in response to the various components of the PI-3 kinase/PTEN pathway. PC3 cells were transiently transfected with 0.5 μg of TOPFLASH reporter together with 2.5 μg of empty vector, ILK-WT, ILK-KD, PTEN-WT, PKB-AAA, or GSK-3-WT. β-Catenin/TCF activity was dramatically reduced due to PTEN, ILK-KD, and GSK-3-WT. The effect of PKB-AAA upon TOPFLASH activity was more modest in comparison. Parallel cotransfections with the various plasmids and FOPFLASH served as a negative control for TOPFLASH activity.

(A) Bar graph represents quantification of GSK-3 kinase activities by densitometric analysis (Odu/mm²) in PC3 cells transiently transfected with empty vector (control), ILK-WT (78%), ILK-KD (79%), PKB-AAA (79%), or PTEN-WT (82%). Values in brackets indicate the transfection efficiency of the various plasmids. Top panel is a representative autoradiograph of GSK-3 kinase activities in the various transfectants. To evaluate stimulation of GSK-3 activity, transfected cells were serum starved for 18 h, refed with serum for 1 h, and then analyzed for GSK-3 kinase activity by using GS-1 peptide as a substrate. Although PTEN and ILK-KD induced a dramatic increase in GSK-3 kinase activity (∼3–4-fold) the effect of PKB-AAA was more modest (∼1.6-fold). GSK-3 kinase activity was determined as described in Materials and Methods. Immunoblot with anti–GSK-3 antibody shows equivalent amounts of GSK-3 in each extract (bottom). (B) Bar graph represents quantification of ILK kinase activity by densitometric analysis (Odu/mm²) in serum-starved (18 h) PC3 cells. ILK, purified by immunoprecipitation with anti-ILK antibody, was coincubated with purified GSK-3-KD and [γ-³²P]ATP in the presence or absence of the ILK inhibitor KP-SD-1. Bottom panel represents an in vitro ILK kinase assay, where recombinant ILK prepared in insect cells was coincubated with GSK-3-KD and ATP in the presence or absence of an ILK inhibitor, KP-SD-1. Phosphorylated GSK-3 was detected by Western blot analysis using anti–GSK-3-Ser-9-P antibody. Odu, optical density units.