Limits...
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.

Show MeSH

Related in: MedlinePlus

In vivo phosphorylation of β-catenin is strongly decreased in V12Ras-overexpressing keratinocytes and mainly affects the P-Ser content. (a) β-catenin was immunoprecipitated from 32P-metabolically labeled Pam212 (P) and PamV12Ras cells from two independent infection assays (Ras1 and Ras2), resolved on polyacrylamide gel and subjected to autoradiography (top) and immunoblotting of the same gel for β-catenin (bottom). The 32P-ratio of β-catenin in the different cell lines was quantitated by densitometric scanning of the bands present in the autoradiogram and immunoblotting. (b) Phosphoamino acid analysis of β-catenin. 32P–β-catenin bands from Pam and Ras2 (Ras) cells were excised from the membranes and processed as described in Materials and Methods (top). Migration of standard amino acids, identified by ninhydrin staining, is indicated at the bottom and by the ovals (top). The TLC plates were simultaneously autoradiographed for 21 d. Only 32P-Ser could be detected, and it was strongly reduced in Ras cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169475&req=5

Figure 6: In vivo phosphorylation of β-catenin is strongly decreased in V12Ras-overexpressing keratinocytes and mainly affects the P-Ser content. (a) β-catenin was immunoprecipitated from 32P-metabolically labeled Pam212 (P) and PamV12Ras cells from two independent infection assays (Ras1 and Ras2), resolved on polyacrylamide gel and subjected to autoradiography (top) and immunoblotting of the same gel for β-catenin (bottom). The 32P-ratio of β-catenin in the different cell lines was quantitated by densitometric scanning of the bands present in the autoradiogram and immunoblotting. (b) Phosphoamino acid analysis of β-catenin. 32P–β-catenin bands from Pam and Ras2 (Ras) cells were excised from the membranes and processed as described in Materials and Methods (top). Migration of standard amino acids, identified by ninhydrin staining, is indicated at the bottom and by the ovals (top). The TLC plates were simultaneously autoradiographed for 21 d. Only 32P-Ser could be detected, and it was strongly reduced in Ras cells.

Mentions: To further analyze the status of β-catenin in control and Ras-transformed keratinocytes, we performed in vivo phosphorylation analysis on both cell types after 4 h of metabolic labeling. The level of β-catenin phosphorylation in PamV12Ras cells resulting from two independent infections, Ras1 and Ras2, was significantly lower than that of parental Pam212 cells (Fig. 6 a). Quantitive analysis of the phosphorylated and total immunoprecipitated β-catenin indicated that the ratio of [32P]β-catenin in Ras1 and Ras2 keratinocytes was 6% and 17% relative to that of the parental cells. In addition, the phosphoamino acid analysis of immunoprecipitated β-catenin showed that P-Ser was the major phosphorylated amino acid, and, as expected, the relative P-Ser content of β-catenin in Ras-transduced cells was much lower than that of parental cells (Fig. 6 b). The P-Tyr content of β-catenin could not be detected in the phosphoamino acid analysis, probably because of the lower stability of P-Tyr residues to acid hydrolysis (Duclos et al. 1991) or to a low level of P-Tyr labeling in the 4-h pulse, in contrast to the steady state levels detected in the immunoprecipitation analysis (Fig. 3 c). These results indicate that activated H-Ras induces cytoplasmic accumulation of hypophosphorylated β-catenin and inhibits its interaction with APC in keratinocytes, through a mechanism apparently independent of stable interactions with or reduced activity of GSK3β.


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)

In vivo phosphorylation of β-catenin is strongly decreased in V12Ras-overexpressing keratinocytes and mainly affects the P-Ser content. (a) β-catenin was immunoprecipitated from 32P-metabolically labeled Pam212 (P) and PamV12Ras cells from two independent infection assays (Ras1 and Ras2), resolved on polyacrylamide gel and subjected to autoradiography (top) and immunoblotting of the same gel for β-catenin (bottom). The 32P-ratio of β-catenin in the different cell lines was quantitated by densitometric scanning of the bands present in the autoradiogram and immunoblotting. (b) Phosphoamino acid analysis of β-catenin. 32P–β-catenin bands from Pam and Ras2 (Ras) cells were excised from the membranes and processed as described in Materials and Methods (top). Migration of standard amino acids, identified by ninhydrin staining, is indicated at the bottom and by the ovals (top). The TLC plates were simultaneously autoradiographed for 21 d. Only 32P-Ser could be detected, and it was strongly reduced in Ras cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169475&req=5

Figure 6: In vivo phosphorylation of β-catenin is strongly decreased in V12Ras-overexpressing keratinocytes and mainly affects the P-Ser content. (a) β-catenin was immunoprecipitated from 32P-metabolically labeled Pam212 (P) and PamV12Ras cells from two independent infection assays (Ras1 and Ras2), resolved on polyacrylamide gel and subjected to autoradiography (top) and immunoblotting of the same gel for β-catenin (bottom). The 32P-ratio of β-catenin in the different cell lines was quantitated by densitometric scanning of the bands present in the autoradiogram and immunoblotting. (b) Phosphoamino acid analysis of β-catenin. 32P–β-catenin bands from Pam and Ras2 (Ras) cells were excised from the membranes and processed as described in Materials and Methods (top). Migration of standard amino acids, identified by ninhydrin staining, is indicated at the bottom and by the ovals (top). The TLC plates were simultaneously autoradiographed for 21 d. Only 32P-Ser could be detected, and it was strongly reduced in Ras cells.
Mentions: To further analyze the status of β-catenin in control and Ras-transformed keratinocytes, we performed in vivo phosphorylation analysis on both cell types after 4 h of metabolic labeling. The level of β-catenin phosphorylation in PamV12Ras cells resulting from two independent infections, Ras1 and Ras2, was significantly lower than that of parental Pam212 cells (Fig. 6 a). Quantitive analysis of the phosphorylated and total immunoprecipitated β-catenin indicated that the ratio of [32P]β-catenin in Ras1 and Ras2 keratinocytes was 6% and 17% relative to that of the parental cells. In addition, the phosphoamino acid analysis of immunoprecipitated β-catenin showed that P-Ser was the major phosphorylated amino acid, and, as expected, the relative P-Ser content of β-catenin in Ras-transduced cells was much lower than that of parental cells (Fig. 6 b). The P-Tyr content of β-catenin could not be detected in the phosphoamino acid analysis, probably because of the lower stability of P-Tyr residues to acid hydrolysis (Duclos et al. 1991) or to a low level of P-Tyr labeling in the 4-h pulse, in contrast to the steady state levels detected in the immunoprecipitation analysis (Fig. 3 c). These results indicate that activated H-Ras induces cytoplasmic accumulation of hypophosphorylated β-catenin and inhibits its interaction with APC in keratinocytes, through a mechanism apparently independent of stable interactions with or reduced activity of GSK3β.

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