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H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

Espada J, Pérez-Moreno M, Braga VM, Rodriguez-Viciana P, Cano A - J. Cell Biol. (1999)

Bottom Line: Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity.In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected.Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28029 Madrid, Spain.

ABSTRACT
The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.

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Inhibition of PI3K activity prevents E-cadherin/catenin complexes disassembly induced by V12Ras. (a) Confluent patches of Pam212 cells were preincubated with the PI3K inhibitor wortmannin (200 nM) for 30 min, microinjected with V12Ras (0.5 μg/μl), and after 3 h, fixed in methanol. Cells were stained for E-cadherin, and microinjected cells were identified by coinjection of dextran–Texas red. (b) Control PamNeo (top, Neo) or PamV12Ras (middle and bottom, Ras) cells were extracted in NT buffer for 15 min on ice, fixed in 3.7% formaldehyde, and stained for E-cadherin (left) or β-catenin, using mouse mAb (right). PamV12Ras cells showed in the bottom were preincubated with the PI3K inhibitor wortmannin (Ras+W) for 1 h before NT buffer extraction. The nuclear staining detected with β-catenin is background staining because of nonspecific binding of the BODIPY-conjugated anti–mouse IgG observed after detergent extraction. Both E-cadherin and β-catenin are redistributed to the detergent soluble fraction in V12Ras-expressing cells and this solubilization is blocked after inhibition of PI3K activity in those cells. Images were obtained in an Axiophot microscope. Bars, 10 μm.
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Figure 2: Inhibition of PI3K activity prevents E-cadherin/catenin complexes disassembly induced by V12Ras. (a) Confluent patches of Pam212 cells were preincubated with the PI3K inhibitor wortmannin (200 nM) for 30 min, microinjected with V12Ras (0.5 μg/μl), and after 3 h, fixed in methanol. Cells were stained for E-cadherin, and microinjected cells were identified by coinjection of dextran–Texas red. (b) Control PamNeo (top, Neo) or PamV12Ras (middle and bottom, Ras) cells were extracted in NT buffer for 15 min on ice, fixed in 3.7% formaldehyde, and stained for E-cadherin (left) or β-catenin, using mouse mAb (right). PamV12Ras cells showed in the bottom were preincubated with the PI3K inhibitor wortmannin (Ras+W) for 1 h before NT buffer extraction. The nuclear staining detected with β-catenin is background staining because of nonspecific binding of the BODIPY-conjugated anti–mouse IgG observed after detergent extraction. Both E-cadherin and β-catenin are redistributed to the detergent soluble fraction in V12Ras-expressing cells and this solubilization is blocked after inhibition of PI3K activity in those cells. Images were obtained in an Axiophot microscope. Bars, 10 μm.

Mentions: To analyze short-term effects of activated H-Ras on the E-cadherin/catenin complexes, we chose the murine epidermal keratinocyte cell line Pam212, obtained after spontaneous immortalization of a primary keratinocyte cell culture (Yuspa et al. 1980). This cell line maintains all the epidermal characteristics of keratinocytes, expresses a normal H-Ras protooncogene, and is nontumorigenic when injected into athymic nude mice (Missero et al. 1991; Sánchez-Prieto et al. 1995). The dominant active form of H-Ras (V12Ras; 0.5 μg/μl) protein was microinjected in confluent Pam212 keratinocytes showing stable cell–cell contacts, and the cells were fixed and stained for different proteins after 1–24 h of incubation. As shown in Fig. 1, Fig. 2Fig. 3Fig. 4 h after microinjection E-cadherin (Fig. 1 b) and α-catenin (Fig. 1 d) were completely absent from the cell–cell contacts established between microinjected cells. At the same time, V12Ras induced a strong cytoplasmic staining of endogenous β-catenin (Fig. 1f and Fig. h). In addition, a diffuse nuclear labeling for β-catenin could be detected in some of the microinjected cells (Fig. 1 h). Microinjection of Pam212 cells with a dominant negative H-Ras protein, N17Ras induced no alterations in the adhesion complexes or β-catenin localization (Fig. 1j and Fig. k). Our data indicate the specificity of V12Ras effects and are in agreement with previous data using anti–Ras antibodies in human keratinocytes (Braga, V.M.M., M. Betson, and N. Lamarche-Vane, manuscript submitted for publication). 16 h after V12Ras microinjection in Pam212 cells, E-cadherin and α-catenin remained absent from the cell–cell contacts and β-catenin was still faintly detected in the cytoplasm (data not shown), indicating that dominant active H-Ras induces dismantling of E-cadherin/catenin complexes and cytoplasmic accumulation of β-catenin in mouse epidermal keratinocytes.


H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

Espada J, Pérez-Moreno M, Braga VM, Rodriguez-Viciana P, Cano A - J. Cell Biol. (1999)

Inhibition of PI3K activity prevents E-cadherin/catenin complexes disassembly induced by V12Ras. (a) Confluent patches of Pam212 cells were preincubated with the PI3K inhibitor wortmannin (200 nM) for 30 min, microinjected with V12Ras (0.5 μg/μl), and after 3 h, fixed in methanol. Cells were stained for E-cadherin, and microinjected cells were identified by coinjection of dextran–Texas red. (b) Control PamNeo (top, Neo) or PamV12Ras (middle and bottom, Ras) cells were extracted in NT buffer for 15 min on ice, fixed in 3.7% formaldehyde, and stained for E-cadherin (left) or β-catenin, using mouse mAb (right). PamV12Ras cells showed in the bottom were preincubated with the PI3K inhibitor wortmannin (Ras+W) for 1 h before NT buffer extraction. The nuclear staining detected with β-catenin is background staining because of nonspecific binding of the BODIPY-conjugated anti–mouse IgG observed after detergent extraction. Both E-cadherin and β-catenin are redistributed to the detergent soluble fraction in V12Ras-expressing cells and this solubilization is blocked after inhibition of PI3K activity in those cells. Images were obtained in an Axiophot microscope. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169475&req=5

Figure 2: Inhibition of PI3K activity prevents E-cadherin/catenin complexes disassembly induced by V12Ras. (a) Confluent patches of Pam212 cells were preincubated with the PI3K inhibitor wortmannin (200 nM) for 30 min, microinjected with V12Ras (0.5 μg/μl), and after 3 h, fixed in methanol. Cells were stained for E-cadherin, and microinjected cells were identified by coinjection of dextran–Texas red. (b) Control PamNeo (top, Neo) or PamV12Ras (middle and bottom, Ras) cells were extracted in NT buffer for 15 min on ice, fixed in 3.7% formaldehyde, and stained for E-cadherin (left) or β-catenin, using mouse mAb (right). PamV12Ras cells showed in the bottom were preincubated with the PI3K inhibitor wortmannin (Ras+W) for 1 h before NT buffer extraction. The nuclear staining detected with β-catenin is background staining because of nonspecific binding of the BODIPY-conjugated anti–mouse IgG observed after detergent extraction. Both E-cadherin and β-catenin are redistributed to the detergent soluble fraction in V12Ras-expressing cells and this solubilization is blocked after inhibition of PI3K activity in those cells. Images were obtained in an Axiophot microscope. Bars, 10 μm.
Mentions: To analyze short-term effects of activated H-Ras on the E-cadherin/catenin complexes, we chose the murine epidermal keratinocyte cell line Pam212, obtained after spontaneous immortalization of a primary keratinocyte cell culture (Yuspa et al. 1980). This cell line maintains all the epidermal characteristics of keratinocytes, expresses a normal H-Ras protooncogene, and is nontumorigenic when injected into athymic nude mice (Missero et al. 1991; Sánchez-Prieto et al. 1995). The dominant active form of H-Ras (V12Ras; 0.5 μg/μl) protein was microinjected in confluent Pam212 keratinocytes showing stable cell–cell contacts, and the cells were fixed and stained for different proteins after 1–24 h of incubation. As shown in Fig. 1, Fig. 2Fig. 3Fig. 4 h after microinjection E-cadherin (Fig. 1 b) and α-catenin (Fig. 1 d) were completely absent from the cell–cell contacts established between microinjected cells. At the same time, V12Ras induced a strong cytoplasmic staining of endogenous β-catenin (Fig. 1f and Fig. h). In addition, a diffuse nuclear labeling for β-catenin could be detected in some of the microinjected cells (Fig. 1 h). Microinjection of Pam212 cells with a dominant negative H-Ras protein, N17Ras induced no alterations in the adhesion complexes or β-catenin localization (Fig. 1j and Fig. k). Our data indicate the specificity of V12Ras effects and are in agreement with previous data using anti–Ras antibodies in human keratinocytes (Braga, V.M.M., M. Betson, and N. Lamarche-Vane, manuscript submitted for publication). 16 h after V12Ras microinjection in Pam212 cells, E-cadherin and α-catenin remained absent from the cell–cell contacts and β-catenin was still faintly detected in the cytoplasm (data not shown), indicating that dominant active H-Ras induces dismantling of E-cadherin/catenin complexes and cytoplasmic accumulation of β-catenin in mouse epidermal keratinocytes.

Bottom Line: Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity.In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected.Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28029 Madrid, Spain.

ABSTRACT
The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.

Show MeSH
Related in: MedlinePlus