Limits...
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.

Show MeSH

Related in: MedlinePlus

Membrane association of APC in HCT116 colon carcinoma cells. (a) Western blot showing distribution of APC after high speed centrifugation. APC present in the postnuclear fraction (PNF) distributes into both the high speed pellet fraction (P100) and the high speed supernatant fraction (S100). Equal proportions of P100 and S100 samples were loaded. (b) Dilution series for measurement of APC by Western blotting. (c) APC floats with membranes in equilibrium density gradients. Graph and corresponding Western blot illustrating distribution of APC, E-cadherin (marker for membranes), and total protein in each fraction after equilibrium density flotation of P100 fractions. (d) Solubility of membrane-bound APC after treatment with detergents or high salt. Membrane-containing fractions after P100 density flotation (fractions 30–33% in panel c) were pooled, treated with 1% NP-40, 1% octyl glucoside (OG) or 1 M NaCl, and subjected to a high speed spin. One third of starting material was loaded in lane M (membrane) and resulting pellet and supernatant fractions were loaded in lanes P and S, respectively. Membrane-bound APC is only partially solubilized by solubilization of membranes with detergents, whereas high salt treatment does not result in the release of APC into the soluble fraction. Moreover, after high salt treatment, APC continues to float with membranes in density gradients (data not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196003&req=5

Figure 2: Membrane association of APC in HCT116 colon carcinoma cells. (a) Western blot showing distribution of APC after high speed centrifugation. APC present in the postnuclear fraction (PNF) distributes into both the high speed pellet fraction (P100) and the high speed supernatant fraction (S100). Equal proportions of P100 and S100 samples were loaded. (b) Dilution series for measurement of APC by Western blotting. (c) APC floats with membranes in equilibrium density gradients. Graph and corresponding Western blot illustrating distribution of APC, E-cadherin (marker for membranes), and total protein in each fraction after equilibrium density flotation of P100 fractions. (d) Solubility of membrane-bound APC after treatment with detergents or high salt. Membrane-containing fractions after P100 density flotation (fractions 30–33% in panel c) were pooled, treated with 1% NP-40, 1% octyl glucoside (OG) or 1 M NaCl, and subjected to a high speed spin. One third of starting material was loaded in lane M (membrane) and resulting pellet and supernatant fractions were loaded in lanes P and S, respectively. Membrane-bound APC is only partially solubilized by solubilization of membranes with detergents, whereas high salt treatment does not result in the release of APC into the soluble fraction. Moreover, after high salt treatment, APC continues to float with membranes in density gradients (data not shown).

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)

Membrane association of APC in HCT116 colon carcinoma cells. (a) Western blot showing distribution of APC after high speed centrifugation. APC present in the postnuclear fraction (PNF) distributes into both the high speed pellet fraction (P100) and the high speed supernatant fraction (S100). Equal proportions of P100 and S100 samples were loaded. (b) Dilution series for measurement of APC by Western blotting. (c) APC floats with membranes in equilibrium density gradients. Graph and corresponding Western blot illustrating distribution of APC, E-cadherin (marker for membranes), and total protein in each fraction after equilibrium density flotation of P100 fractions. (d) Solubility of membrane-bound APC after treatment with detergents or high salt. Membrane-containing fractions after P100 density flotation (fractions 30–33% in panel c) were pooled, treated with 1% NP-40, 1% octyl glucoside (OG) or 1 M NaCl, and subjected to a high speed spin. One third of starting material was loaded in lane M (membrane) and resulting pellet and supernatant fractions were loaded in lanes P and S, respectively. Membrane-bound APC is only partially solubilized by solubilization of membranes with detergents, whereas high salt treatment does not result in the release of APC into the soluble fraction. Moreover, after high salt treatment, APC continues to float with membranes in density gradients (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Membrane association of APC in HCT116 colon carcinoma cells. (a) Western blot showing distribution of APC after high speed centrifugation. APC present in the postnuclear fraction (PNF) distributes into both the high speed pellet fraction (P100) and the high speed supernatant fraction (S100). Equal proportions of P100 and S100 samples were loaded. (b) Dilution series for measurement of APC by Western blotting. (c) APC floats with membranes in equilibrium density gradients. Graph and corresponding Western blot illustrating distribution of APC, E-cadherin (marker for membranes), and total protein in each fraction after equilibrium density flotation of P100 fractions. (d) Solubility of membrane-bound APC after treatment with detergents or high salt. Membrane-containing fractions after P100 density flotation (fractions 30–33% in panel c) were pooled, treated with 1% NP-40, 1% octyl glucoside (OG) or 1 M NaCl, and subjected to a high speed spin. One third of starting material was loaded in lane M (membrane) and resulting pellet and supernatant fractions were loaded in lanes P and S, respectively. Membrane-bound APC is only partially solubilized by solubilization of membranes with detergents, whereas high salt treatment does not result in the release of APC into the soluble fraction. Moreover, after high salt treatment, APC continues to float with membranes in density gradients (data not shown).
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