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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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Schematic representation of fractionation protocol used to analyze subcellular distribution of APC and components of the β-catenin destruction complex in HCT116 and MCF-7 cells. Cells were lysed in hypotonic lysis buffer without detergents (w/o det.) and homogenized using a Dounce homogenizer. Unbroken cells and nuclei were removed by low speed centrifugation (5,000 rpm for 30 min). Postnuclear supernatants were further fractionated by high speed centrifugation (100,000 g for 1 h) into a pellet fraction (P100, >90S) and a supernatant fraction (S100, <90S). Membrane association of proteins was analyzed by sucrose density equilibrium flotation of P100 fractions (density of membranes ∼1.13 g/cm3; density of dense fractions ∼1.3 g/cm3). The S100 sample was further fractionated by velocity sedimentation. Size fractions of ∼4–5S, ∼20S, and ∼60S were distinguished based on distribution of total protein, APC, and axin.
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Figure 1: Schematic representation of fractionation protocol used to analyze subcellular distribution of APC and components of the β-catenin destruction complex in HCT116 and MCF-7 cells. Cells were lysed in hypotonic lysis buffer without detergents (w/o det.) and homogenized using a Dounce homogenizer. Unbroken cells and nuclei were removed by low speed centrifugation (5,000 rpm for 30 min). Postnuclear supernatants were further fractionated by high speed centrifugation (100,000 g for 1 h) into a pellet fraction (P100, >90S) and a supernatant fraction (S100, <90S). Membrane association of proteins was analyzed by sucrose density equilibrium flotation of P100 fractions (density of membranes ∼1.13 g/cm3; density of dense fractions ∼1.3 g/cm3). The S100 sample was further fractionated by velocity sedimentation. Size fractions of ∼4–5S, ∼20S, and ∼60S were distinguished based on distribution of total protein, APC, and axin.

Mentions: To localize APC at the subcellular level, we initially used the human colon carcinoma cell line HCT116, because this cell line expresses wild-type APC. It does express a mutant β-catenin protein that carries a deletion of ser45 (Rubinfeld et al. 1997). The fractionation protocol described in Fig. 1 was used. As shown in Fig. 2 a, APC distributed approximately equally into pelletable and soluble material after the initial 100,000-g spin. This distribution pattern was consistently observed when fully confluent monolayers were used.


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)

Schematic representation of fractionation protocol used to analyze subcellular distribution of APC and components of the β-catenin destruction complex in HCT116 and MCF-7 cells. Cells were lysed in hypotonic lysis buffer without detergents (w/o det.) and homogenized using a Dounce homogenizer. Unbroken cells and nuclei were removed by low speed centrifugation (5,000 rpm for 30 min). Postnuclear supernatants were further fractionated by high speed centrifugation (100,000 g for 1 h) into a pellet fraction (P100, >90S) and a supernatant fraction (S100, <90S). Membrane association of proteins was analyzed by sucrose density equilibrium flotation of P100 fractions (density of membranes ∼1.13 g/cm3; density of dense fractions ∼1.3 g/cm3). The S100 sample was further fractionated by velocity sedimentation. Size fractions of ∼4–5S, ∼20S, and ∼60S were distinguished based on distribution of total protein, APC, and axin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic representation of fractionation protocol used to analyze subcellular distribution of APC and components of the β-catenin destruction complex in HCT116 and MCF-7 cells. Cells were lysed in hypotonic lysis buffer without detergents (w/o det.) and homogenized using a Dounce homogenizer. Unbroken cells and nuclei were removed by low speed centrifugation (5,000 rpm for 30 min). Postnuclear supernatants were further fractionated by high speed centrifugation (100,000 g for 1 h) into a pellet fraction (P100, >90S) and a supernatant fraction (S100, <90S). Membrane association of proteins was analyzed by sucrose density equilibrium flotation of P100 fractions (density of membranes ∼1.13 g/cm3; density of dense fractions ∼1.3 g/cm3). The S100 sample was further fractionated by velocity sedimentation. Size fractions of ∼4–5S, ∼20S, and ∼60S were distinguished based on distribution of total protein, APC, and axin.
Mentions: To localize APC at the subcellular level, we initially used the human colon carcinoma cell line HCT116, because this cell line expresses wild-type APC. It does express a mutant β-catenin protein that carries a deletion of ser45 (Rubinfeld et al. 1997). The fractionation protocol described in Fig. 1 was used. As shown in Fig. 2 a, APC distributed approximately equally into pelletable and soluble material after the initial 100,000-g spin. This distribution pattern was consistently observed when fully confluent monolayers were used.

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