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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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Stable expression of V12Ras in Pam212 keratinocytes inhibits β-catenin–APC interaction, but does not induce stable interaction of β-catenin with GSK3β or affect GSK3β activity. (a) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti-APC antibodies and subjected to immunoblotting for β-catenin (bottom, IP: APC). As a control of the APC level, the extracts were immunoblotted with anti-APC antibodies (top, lysates). (b) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti–β-catenin (mouse mAb) (middle, IP: β-catenin) or anti-GSK3β (bottom, IP: GSK3β) and subjected to immunoblotting for β-catenin, GSK3β, and P-Ser9GSK3β, as indicated. As a control, total cell extracts from Neo and Ras cells (top, lysates) were also included in the immunoblottings. (c) GSK3β activity was assayed in crude cell extracts of control PamNeo (Neo) and PamV12Ras cells from three independent infections (Ras1, Ras2, and Ras3) in the presence and absence of 50 mM lithium chloride using the specific peptide GSM as substrate. The specific activity is represented as the percentage of the difference between the values obtained in the absence and presence of lithium chloride in each cell line. Samples were analyzed in duplicate, and the results show the average value and SD. (d) Lef-1/Tcf–dependent transcriptional activity was measured in control PamNeo (Neo) and PamV12Ras (Ras) cells using pTOPFLASH (Top) and pFOPFLASH (Fop) reporter vectors containing multimerized wild-type (Top) or mutated (Fop) Lef-1/Tcf binding sites. Assays were performed in duplicate samples, and the results show the average value and SD.
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Figure 5: Stable expression of V12Ras in Pam212 keratinocytes inhibits β-catenin–APC interaction, but does not induce stable interaction of β-catenin with GSK3β or affect GSK3β activity. (a) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti-APC antibodies and subjected to immunoblotting for β-catenin (bottom, IP: APC). As a control of the APC level, the extracts were immunoblotted with anti-APC antibodies (top, lysates). (b) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti–β-catenin (mouse mAb) (middle, IP: β-catenin) or anti-GSK3β (bottom, IP: GSK3β) and subjected to immunoblotting for β-catenin, GSK3β, and P-Ser9GSK3β, as indicated. As a control, total cell extracts from Neo and Ras cells (top, lysates) were also included in the immunoblottings. (c) GSK3β activity was assayed in crude cell extracts of control PamNeo (Neo) and PamV12Ras cells from three independent infections (Ras1, Ras2, and Ras3) in the presence and absence of 50 mM lithium chloride using the specific peptide GSM as substrate. The specific activity is represented as the percentage of the difference between the values obtained in the absence and presence of lithium chloride in each cell line. Samples were analyzed in duplicate, and the results show the average value and SD. (d) Lef-1/Tcf–dependent transcriptional activity was measured in control PamNeo (Neo) and PamV12Ras (Ras) cells using pTOPFLASH (Top) and pFOPFLASH (Fop) reporter vectors containing multimerized wild-type (Top) or mutated (Fop) Lef-1/Tcf binding sites. Assays were performed in duplicate samples, and the results show the average value and SD.

Mentions: The above results prompted us to examine the interaction of β-catenin with other known partners in Neo versus Ras cells. Because of the relevance of the β-catenin–APC interaction in the regulation of cytoplasmic β-catenin levels, we investigated whether activated H-Ras could influence such interaction. High levels of soluble β-catenin coprecipitated with APC in control Neo cells (Fig. 5 a, IP: APC, lane Neo), whereas very low levels of β-catenin were detected in the APC immunocomplexes of PamV12Ras cells (Fig. 5 a, IP: APC, Ras lane), even though the level of APC was similar in both cell types (Fig. 5 a, lysates). On the other hand, similar levels of GSK3β protein were detected in whole cell extracts from control Neo and Ras cells (Fig. 5 b, lysates), but no stable association of β-catenin with GSK3β was found in either cell line (Fig. 5 b, IP β-catenin or 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)

Stable expression of V12Ras in Pam212 keratinocytes inhibits β-catenin–APC interaction, but does not induce stable interaction of β-catenin with GSK3β or affect GSK3β activity. (a) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti-APC antibodies and subjected to immunoblotting for β-catenin (bottom, IP: APC). As a control of the APC level, the extracts were immunoblotted with anti-APC antibodies (top, lysates). (b) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti–β-catenin (mouse mAb) (middle, IP: β-catenin) or anti-GSK3β (bottom, IP: GSK3β) and subjected to immunoblotting for β-catenin, GSK3β, and P-Ser9GSK3β, as indicated. As a control, total cell extracts from Neo and Ras cells (top, lysates) were also included in the immunoblottings. (c) GSK3β activity was assayed in crude cell extracts of control PamNeo (Neo) and PamV12Ras cells from three independent infections (Ras1, Ras2, and Ras3) in the presence and absence of 50 mM lithium chloride using the specific peptide GSM as substrate. The specific activity is represented as the percentage of the difference between the values obtained in the absence and presence of lithium chloride in each cell line. Samples were analyzed in duplicate, and the results show the average value and SD. (d) Lef-1/Tcf–dependent transcriptional activity was measured in control PamNeo (Neo) and PamV12Ras (Ras) cells using pTOPFLASH (Top) and pFOPFLASH (Fop) reporter vectors containing multimerized wild-type (Top) or mutated (Fop) Lef-1/Tcf binding sites. Assays were performed in duplicate samples, and the results show the average value and SD.
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Related In: Results  -  Collection

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Figure 5: Stable expression of V12Ras in Pam212 keratinocytes inhibits β-catenin–APC interaction, but does not induce stable interaction of β-catenin with GSK3β or affect GSK3β activity. (a) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti-APC antibodies and subjected to immunoblotting for β-catenin (bottom, IP: APC). As a control of the APC level, the extracts were immunoblotted with anti-APC antibodies (top, lysates). (b) Soluble extracts from PamNeo (Neo) and PamV12Ras (Ras) cells were immunoprecipitated with anti–β-catenin (mouse mAb) (middle, IP: β-catenin) or anti-GSK3β (bottom, IP: GSK3β) and subjected to immunoblotting for β-catenin, GSK3β, and P-Ser9GSK3β, as indicated. As a control, total cell extracts from Neo and Ras cells (top, lysates) were also included in the immunoblottings. (c) GSK3β activity was assayed in crude cell extracts of control PamNeo (Neo) and PamV12Ras cells from three independent infections (Ras1, Ras2, and Ras3) in the presence and absence of 50 mM lithium chloride using the specific peptide GSM as substrate. The specific activity is represented as the percentage of the difference between the values obtained in the absence and presence of lithium chloride in each cell line. Samples were analyzed in duplicate, and the results show the average value and SD. (d) Lef-1/Tcf–dependent transcriptional activity was measured in control PamNeo (Neo) and PamV12Ras (Ras) cells using pTOPFLASH (Top) and pFOPFLASH (Fop) reporter vectors containing multimerized wild-type (Top) or mutated (Fop) Lef-1/Tcf binding sites. Assays were performed in duplicate samples, and the results show the average value and SD.
Mentions: The above results prompted us to examine the interaction of β-catenin with other known partners in Neo versus Ras cells. Because of the relevance of the β-catenin–APC interaction in the regulation of cytoplasmic β-catenin levels, we investigated whether activated H-Ras could influence such interaction. High levels of soluble β-catenin coprecipitated with APC in control Neo cells (Fig. 5 a, IP: APC, lane Neo), whereas very low levels of β-catenin were detected in the APC immunocomplexes of PamV12Ras cells (Fig. 5 a, IP: APC, Ras lane), even though the level of APC was similar in both cell types (Fig. 5 a, lysates). On the other hand, similar levels of GSK3β protein were detected in whole cell extracts from control Neo and Ras cells (Fig. 5 b, lysates), but no stable association of β-catenin with GSK3β was found in either cell line (Fig. 5 b, IP β-catenin or 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