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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.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.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.

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 HCT116 cells. Cells were fractionated according to the scheme in Fig. 1 and analyzed by Western blotting for APC, β-catenin (β-Cat), axin, dishevelled (Dvl), and GSK-3β (GSK). (a) Distribution of proteins into pellet (P100) and supernatant (S100) after high speed centrifugation. Equal proportions of P100 and S100 were loaded. Note that axin, dishevelled, and GSK are mostly cytosolic. (b) Distribution of proteins of the destruction complex and β-catenin after density equilibrium 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 and β-catenin after velocity sizing of S100 fractions. APC distributes into two high molecular weight pools of ∼20S and 60S.
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Figure 9: Distinct fractionation pattern of components of the β-catenin destruction complex in HCT116 cells. Cells were fractionated according to the scheme in Fig. 1 and analyzed by Western blotting for APC, β-catenin (β-Cat), axin, dishevelled (Dvl), and GSK-3β (GSK). (a) Distribution of proteins into pellet (P100) and supernatant (S100) after high speed centrifugation. Equal proportions of P100 and S100 were loaded. Note that axin, dishevelled, and GSK are mostly cytosolic. (b) Distribution of proteins of the destruction complex and β-catenin after density equilibrium 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 and β-catenin after velocity sizing of S100 fractions. APC distributes into two high molecular weight pools of ∼20S and 60S.

Mentions: Although most of the APC localized to the apical membrane in epithelial cells by immunofluorescence, we did identify a soluble pool of APC biochemically upon initial high speed centrifugation (Fig. 2 a). To characterize the soluble fraction of APC further, we determined the size of APC-containing protein complexes by velocity gradient centrifugation. With this method, APC sedimented in two size-fractions corresponding to sedimentation coefficients of ∼20S and 60S (Fig. 9 c and 10 c). In particular, the 60S pool of APC fractionated away from most of the total protein in the S100 fraction, most of which sedimented at a smaller size of ∼4–5S and some at ∼20S (data not shown). This distribution pattern of APC into two distinct pools was repeatedly observed in multiple experiments.


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 HCT116 cells. Cells were fractionated according to the scheme in Fig. 1 and analyzed by Western blotting for APC, β-catenin (β-Cat), axin, dishevelled (Dvl), and GSK-3β (GSK). (a) Distribution of proteins into pellet (P100) and supernatant (S100) after high speed centrifugation. Equal proportions of P100 and S100 were loaded. Note that axin, dishevelled, and GSK are mostly cytosolic. (b) Distribution of proteins of the destruction complex and β-catenin after density equilibrium 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 and β-catenin after velocity sizing of S100 fractions. APC distributes into two high molecular weight pools of ∼20S and 60S.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196003&req=5

Figure 9: Distinct fractionation pattern of components of the β-catenin destruction complex in HCT116 cells. Cells were fractionated according to the scheme in Fig. 1 and analyzed by Western blotting for APC, β-catenin (β-Cat), axin, dishevelled (Dvl), and GSK-3β (GSK). (a) Distribution of proteins into pellet (P100) and supernatant (S100) after high speed centrifugation. Equal proportions of P100 and S100 were loaded. Note that axin, dishevelled, and GSK are mostly cytosolic. (b) Distribution of proteins of the destruction complex and β-catenin after density equilibrium 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 and β-catenin after velocity sizing of S100 fractions. APC distributes into two high molecular weight pools of ∼20S and 60S.
Mentions: Although most of the APC localized to the apical membrane in epithelial cells by immunofluorescence, we did identify a soluble pool of APC biochemically upon initial high speed centrifugation (Fig. 2 a). To characterize the soluble fraction of APC further, we determined the size of APC-containing protein complexes by velocity gradient centrifugation. With this method, APC sedimented in two size-fractions corresponding to sedimentation coefficients of ∼20S and 60S (Fig. 9 c and 10 c). In particular, the 60S pool of APC fractionated away from most of the total protein in the S100 fraction, most of which sedimented at a smaller size of ∼4–5S and some at ∼20S (data not shown). This distribution pattern of APC into two distinct pools was repeatedly observed in multiple experiments.

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.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.Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex.

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