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Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells.

Reinacher-Schick A, Gumbiner BM - J. Cell Biol. (2001)

Bottom Line: Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

ABSTRACT
The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

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Membrane association of APC in MCF-7 breast epithelial cancer cells. (a) Western blot for APC illustrating distribution of APC after fractionation of postnuclear fractions (PNF) into a high speed pellet fraction (P100) and a high speed supernatant fraction (S100). Equal proportions of P100 and S100 were loaded. (b) APC in MCF-7 cells fractionates with membranes in equilibrium density gradients. Graph illustrating relative distribution of total protein and APC in each fraction after density flotation of P100 samples. Corresponding Western blot for APC is shown below.
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Figure 4: Membrane association of APC in MCF-7 breast epithelial cancer cells. (a) Western blot for APC illustrating distribution of APC after fractionation of postnuclear fractions (PNF) into a high speed pellet fraction (P100) and a high speed supernatant fraction (S100). Equal proportions of P100 and S100 were loaded. (b) APC in MCF-7 cells fractionates with membranes in equilibrium density gradients. Graph illustrating relative distribution of total protein and APC in each fraction after density flotation of P100 samples. Corresponding Western blot for APC is shown below.

Mentions: To determine whether the apical membrane localization of APC is unique to the HCT116 colon carcinoma cell line or due to the presence of a mutant β-catenin found in HCT116 cells, we analyzed the breast cancer cell line MCF-7, which expresses wild-type β-catenin and wild-type APC. The distribution of APC during the initial 100,000-g spin was similar in MCF-7 cells compared with HCT116 cells. APC fractionated to about equal proportions into pellet and supernatant (Fig. 4 a). Furthermore, as found in HCT116 cells, APC in MCF-7 cells also sedimented at low densities characteristic of plasma membranes during equilibrium centrifugation, suggesting that APC similarly associates with the plasma membrane in this cell line (Fig. 4 b). To assess whether APC also localizes specifically to the apical plasma membrane in MCF-7 cells, we analyzed this cell line by confocal immunofluorescence microscopy (Fig. 5, a–g). APC localized predominantly to the apical membrane and in general did not overlap with β-catenin, which mainly localized to lateral cell borders (Fig. 5, d–g; negative controls for MCF-7 cells shown in a–c). Also, to evaluate APC localization in a nontransformed epithelial cell line, we examined fully confluent, well-polarized MDCK cells by immunofluorescence microscopy (Fig. 5, k–n; negative controls for MDCK cells shown in h–j). APC also localized apically in MDCK cells, whereas β-catenin was found laterally. In general, no overlap between the two proteins was observed.


Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells.

Reinacher-Schick A, Gumbiner BM - J. Cell Biol. (2001)

Membrane association of APC in MCF-7 breast epithelial cancer cells. (a) Western blot for APC illustrating distribution of APC after fractionation of postnuclear fractions (PNF) into a high speed pellet fraction (P100) and a high speed supernatant fraction (S100). Equal proportions of P100 and S100 were loaded. (b) APC in MCF-7 cells fractionates with membranes in equilibrium density gradients. Graph illustrating relative distribution of total protein and APC in each fraction after density flotation of P100 samples. Corresponding Western blot for APC is shown below.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196003&req=5

Figure 4: Membrane association of APC in MCF-7 breast epithelial cancer cells. (a) Western blot for APC illustrating distribution of APC after fractionation of postnuclear fractions (PNF) into a high speed pellet fraction (P100) and a high speed supernatant fraction (S100). Equal proportions of P100 and S100 were loaded. (b) APC in MCF-7 cells fractionates with membranes in equilibrium density gradients. Graph illustrating relative distribution of total protein and APC in each fraction after density flotation of P100 samples. Corresponding Western blot for APC is shown below.
Mentions: To determine whether the apical membrane localization of APC is unique to the HCT116 colon carcinoma cell line or due to the presence of a mutant β-catenin found in HCT116 cells, we analyzed the breast cancer cell line MCF-7, which expresses wild-type β-catenin and wild-type APC. The distribution of APC during the initial 100,000-g spin was similar in MCF-7 cells compared with HCT116 cells. APC fractionated to about equal proportions into pellet and supernatant (Fig. 4 a). Furthermore, as found in HCT116 cells, APC in MCF-7 cells also sedimented at low densities characteristic of plasma membranes during equilibrium centrifugation, suggesting that APC similarly associates with the plasma membrane in this cell line (Fig. 4 b). To assess whether APC also localizes specifically to the apical plasma membrane in MCF-7 cells, we analyzed this cell line by confocal immunofluorescence microscopy (Fig. 5, a–g). APC localized predominantly to the apical membrane and in general did not overlap with β-catenin, which mainly localized to lateral cell borders (Fig. 5, d–g; negative controls for MCF-7 cells shown in a–c). Also, to evaluate APC localization in a nontransformed epithelial cell line, we examined fully confluent, well-polarized MDCK cells by immunofluorescence microscopy (Fig. 5, k–n; negative controls for MDCK cells shown in h–j). APC also localized apically in MDCK cells, whereas β-catenin was found laterally. In general, no overlap between the two proteins was observed.

Bottom Line: Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

ABSTRACT
The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.

Show MeSH
Related in: MedlinePlus