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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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Distinct fractionation pattern of components of the β-catenin destruction complex in MCF-7 breast cancer cells. Cells were fractionated according to the scheme in Fig. 1 and fractions were analyzed by Western blotting for APC, β-catenin, axin, dishevelled, and GSK-3β. (a) Distribution of proteins into high speed pellet (P100) and high speed supernatant (S100) fractions. Equal proportions of the fractions were loaded. Note that axin, dishevelled, and GSK-3β are predominantly cytosolic. (b) Distribution of components of the destruction complex and β-catenin after density flotation of P100 fractions. Note that only a small fraction of total axin, dishevelled, and GSK-3β is analyzed by P100 flotation since these components are mostly cytosolic. (c) Distribution of components of the destruction complex after velocity centrifugation of S100 fractions. Similar to HCT116 cells, APC distributes into two high molecular weight peaks of ∼20S and ∼60S in MCF-7 cells.
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Figure 10: Distinct fractionation pattern of components of the β-catenin destruction complex in MCF-7 breast cancer cells. Cells were fractionated according to the scheme in Fig. 1 and fractions were analyzed by Western blotting for APC, β-catenin, axin, dishevelled, and GSK-3β. (a) Distribution of proteins into high speed pellet (P100) and high speed supernatant (S100) fractions. Equal proportions of the fractions were loaded. Note that axin, dishevelled, and GSK-3β are predominantly cytosolic. (b) Distribution of components of the destruction complex and β-catenin after density flotation of P100 fractions. Note that only a small fraction of total axin, dishevelled, and GSK-3β is analyzed by P100 flotation since these components are mostly cytosolic. (c) Distribution of components of the destruction complex after velocity centrifugation of S100 fractions. Similar to HCT116 cells, APC distributes into two high molecular weight peaks of ∼20S and ∼60S in MCF-7 cells.

Mentions: To assess whether the distributions of these proteins were unique to HCT116 cells or dependent on the NH2-terminal mutation of β-catenin expressed by this cell line, we also analyzed their fractionation patterns in the breast cancer cell line MCF-7 (Fig. 10). APC as well as β-catenin, axin, dishevelled, and GSK-3β distributed into subcellular pools in MCF-7 cells very similar to HCT116 cells. APC fractionated equally into P100 and S100 pools, whereas axin, dishevelled, and GSK-3β were largely soluble and β-catenin mostly pelleted (Fig. 10 a). In density flotation gradients, most complex components from MCF-7 cells behaved comparable to those from HCT116 cells (Fig. 10 b). APC and β-catenin mostly floated with membranes, whereas the small amounts of particulate dishevelled were mainly detected in dense fractions. In contrast to the HCT116 cells where the small P100 pools of axin and GSK-3β distributed in both membrane and dense fractions, the small pool of particulate axin fractionated exclusively in membrane fractions, and most of the pelletable GSK-3β sedimented in the dense fractions. Nonetheless, because the fraction of axin and GSK-3β present in the P100 before flotation is minor compared with the soluble pool of these proteins (see Fig. 10 a), their overall distributions are very similar in HCT116 and MCF-7 cells.


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)

Distinct fractionation pattern of components of the β-catenin destruction complex in MCF-7 breast cancer cells. Cells were fractionated according to the scheme in Fig. 1 and fractions were analyzed by Western blotting for APC, β-catenin, axin, dishevelled, and GSK-3β. (a) Distribution of proteins into high speed pellet (P100) and high speed supernatant (S100) fractions. Equal proportions of the fractions were loaded. Note that axin, dishevelled, and GSK-3β are predominantly cytosolic. (b) Distribution of components of the destruction complex and β-catenin after density flotation of P100 fractions. Note that only a small fraction of total axin, dishevelled, and GSK-3β is analyzed by P100 flotation since these components are mostly cytosolic. (c) Distribution of components of the destruction complex after velocity centrifugation of S100 fractions. Similar to HCT116 cells, APC distributes into two high molecular weight peaks of ∼20S and ∼60S in MCF-7 cells.
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Related In: Results  -  Collection

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

Figure 10: Distinct fractionation pattern of components of the β-catenin destruction complex in MCF-7 breast cancer cells. Cells were fractionated according to the scheme in Fig. 1 and fractions were analyzed by Western blotting for APC, β-catenin, axin, dishevelled, and GSK-3β. (a) Distribution of proteins into high speed pellet (P100) and high speed supernatant (S100) fractions. Equal proportions of the fractions were loaded. Note that axin, dishevelled, and GSK-3β are predominantly cytosolic. (b) Distribution of components of the destruction complex and β-catenin after density flotation of P100 fractions. Note that only a small fraction of total axin, dishevelled, and GSK-3β is analyzed by P100 flotation since these components are mostly cytosolic. (c) Distribution of components of the destruction complex after velocity centrifugation of S100 fractions. Similar to HCT116 cells, APC distributes into two high molecular weight peaks of ∼20S and ∼60S in MCF-7 cells.
Mentions: To assess whether the distributions of these proteins were unique to HCT116 cells or dependent on the NH2-terminal mutation of β-catenin expressed by this cell line, we also analyzed their fractionation patterns in the breast cancer cell line MCF-7 (Fig. 10). APC as well as β-catenin, axin, dishevelled, and GSK-3β distributed into subcellular pools in MCF-7 cells very similar to HCT116 cells. APC fractionated equally into P100 and S100 pools, whereas axin, dishevelled, and GSK-3β were largely soluble and β-catenin mostly pelleted (Fig. 10 a). In density flotation gradients, most complex components from MCF-7 cells behaved comparable to those from HCT116 cells (Fig. 10 b). APC and β-catenin mostly floated with membranes, whereas the small amounts of particulate dishevelled were mainly detected in dense fractions. In contrast to the HCT116 cells where the small P100 pools of axin and GSK-3β distributed in both membrane and dense fractions, the small pool of particulate axin fractionated exclusively in membrane fractions, and most of the pelletable GSK-3β sedimented in the dense fractions. Nonetheless, because the fraction of axin and GSK-3β present in the P100 before flotation is minor compared with the soluble pool of these proteins (see Fig. 10 a), their overall distributions are very similar in HCT116 and MCF-7 cells.

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