Limits...
Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

Persad S, Troussard AA, McPhee TR, Mulholland DJ, Dedhar S - J. Cell Biol. (2001)

Bottom Line: We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN.TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity.Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

View Article: PubMed Central - PubMed

Affiliation: British Columbia Cancer Agency, Jack Bell Research Center, Vancouver V6H 3Z6, British Columbia, Canada.

ABSTRACT
beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Show MeSH

Related in: MedlinePlus

(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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192018&req=5

Figure 4: (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.

Mentions: To further understand the relative roles of ILK and PTEN in the GSK-3–mediated regulation of β-catenin, we evaluated GSK-3 activity in PC3 cells transiently transfected with ILK-WT, ILK-KD, PKB-AAA, or PTEN-WT. As shown in Fig. 4 A, although both PTEN-WT and dominant negative ILK-KD induced a significant increase in GSK-3 activity (∼3–4-fold) the effect of dominant negative PKB-AAA was lower (∼1.6-fold). This difference is unlikely to be due to differences in transfection efficiencies, since all three plasmids had comparable transfection efficiencies (see legend to Fig. 4). Transfection of ILK-WT cDNA did not alter the activity of GSK-3 compared with the control. These data confirm that GSK-3 activity, which is known to be involved in controlling β-catenin levels in cells (Morin 1999; Behrens 2000), is regulated to various extents by PTEN, ILK, and PKB. Although both ILK (Delcommenne et al. 1998; Troussard et al. 1999) and PKB (Cross et al. 1995) are known to phosphorylate GSK-3, the greater stimulatory effect of PTEN and ILK-KD upon GSK-3 activity compared with PKB-AAA indicate that in these cells, GSK-3 is largely regulated by PTEN in an ILK-dependent manner. The critical protein in the regulation of β-catenin phosphorylation and stability is GSK-3, whose activity is also regulated by phosphorylation at serine 9. Although PKB can phosphorylate GSK-3 at this site and inhibit its activity, our results also implicate a direct role for ILK in the regulation of GSK-3 activity. To determine if ILK can directly phosphorylate GSK-3, we coincubated ILK purified from serum-starved PC3 cells by immunoprecipitation with anti-ILK antibody with purified GSK-3-KD in the presence of [γ-32P]ATP and kinase reaction buffer. As shown in Fig. 4 B, top, GSK-3 was successfully phosphorylated by the immunoprecipitated ILK and this phosphorylation was significantly inhibited in the presence of the ILK inhibitor KP-SD-1 (Persad et al. 2001), demonstrating the specificity of the phosphorylation of GSK-3 by ILK. Although it is conceivable that PKB, which may have coimmunoprecipitated along with ILK, is responsible for the GSK-3 phosphorylation, the inhibitory effect of the ILK inhibitor, KP-SD-1 argues against this. KP-SD-1 is ineffective in inhibiting the activity of purified PKB in vitro (Persad et al. 2001). This is further supported by an in vitro kinase assay that shows that ILK can directly phosphorylate GSK-3 at serine 9 in the absence of PKB. This was done by using recombinant ILK protein prepared in insect cells, purified GSK-3-KD as the substrate, and ATP as the phosphate group donor, followed by Western blotting with anti–GSK-3-Ser-9-P antibody. As shown in Fig. 4 B, bottom, ILK can indeed promote the direct phosphorylation of GSK-3 on serine 9 and this phosphorylation is inhibited in the presence of the ILK inhibitor, KP-SD-1.


Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

Persad S, Troussard AA, McPhee TR, Mulholland DJ, Dedhar S - J. Cell Biol. (2001)

(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192018&req=5

Figure 4: (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.
Mentions: To further understand the relative roles of ILK and PTEN in the GSK-3–mediated regulation of β-catenin, we evaluated GSK-3 activity in PC3 cells transiently transfected with ILK-WT, ILK-KD, PKB-AAA, or PTEN-WT. As shown in Fig. 4 A, although both PTEN-WT and dominant negative ILK-KD induced a significant increase in GSK-3 activity (∼3–4-fold) the effect of dominant negative PKB-AAA was lower (∼1.6-fold). This difference is unlikely to be due to differences in transfection efficiencies, since all three plasmids had comparable transfection efficiencies (see legend to Fig. 4). Transfection of ILK-WT cDNA did not alter the activity of GSK-3 compared with the control. These data confirm that GSK-3 activity, which is known to be involved in controlling β-catenin levels in cells (Morin 1999; Behrens 2000), is regulated to various extents by PTEN, ILK, and PKB. Although both ILK (Delcommenne et al. 1998; Troussard et al. 1999) and PKB (Cross et al. 1995) are known to phosphorylate GSK-3, the greater stimulatory effect of PTEN and ILK-KD upon GSK-3 activity compared with PKB-AAA indicate that in these cells, GSK-3 is largely regulated by PTEN in an ILK-dependent manner. The critical protein in the regulation of β-catenin phosphorylation and stability is GSK-3, whose activity is also regulated by phosphorylation at serine 9. Although PKB can phosphorylate GSK-3 at this site and inhibit its activity, our results also implicate a direct role for ILK in the regulation of GSK-3 activity. To determine if ILK can directly phosphorylate GSK-3, we coincubated ILK purified from serum-starved PC3 cells by immunoprecipitation with anti-ILK antibody with purified GSK-3-KD in the presence of [γ-32P]ATP and kinase reaction buffer. As shown in Fig. 4 B, top, GSK-3 was successfully phosphorylated by the immunoprecipitated ILK and this phosphorylation was significantly inhibited in the presence of the ILK inhibitor KP-SD-1 (Persad et al. 2001), demonstrating the specificity of the phosphorylation of GSK-3 by ILK. Although it is conceivable that PKB, which may have coimmunoprecipitated along with ILK, is responsible for the GSK-3 phosphorylation, the inhibitory effect of the ILK inhibitor, KP-SD-1 argues against this. KP-SD-1 is ineffective in inhibiting the activity of purified PKB in vitro (Persad et al. 2001). This is further supported by an in vitro kinase assay that shows that ILK can directly phosphorylate GSK-3 at serine 9 in the absence of PKB. This was done by using recombinant ILK protein prepared in insect cells, purified GSK-3-KD as the substrate, and ATP as the phosphate group donor, followed by Western blotting with anti–GSK-3-Ser-9-P antibody. As shown in Fig. 4 B, bottom, ILK can indeed promote the direct phosphorylation of GSK-3 on serine 9 and this phosphorylation is inhibited in the presence of the ILK inhibitor, KP-SD-1.

Bottom Line: We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN.TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity.Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

View Article: PubMed Central - PubMed

Affiliation: British Columbia Cancer Agency, Jack Bell Research Center, Vancouver V6H 3Z6, British Columbia, Canada.

ABSTRACT
beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Show MeSH
Related in: MedlinePlus