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

Localization of mutant forms of APC to the apical membrane in colon cancer cell lines. (a) Linear representation of the full-length human APC protein. Several known motifs are shown on top, including the oligomerization domain (oligom.), armadillo repeats (arm. repeats), the 15– and 20–amino acid repeats (both known to bind β-catenin), basic domain, and Dlg binding site. Regions for axin binding are shown below. Immunolocalization of APC (red) and β-catenin (green) in DLD-1 (b–e), LoVo (f–i), and LS411 (j–m) cells. Linear representation of truncated mutant forms of the APC protein expressed by the respective cell line is also shown. Images of successive sections of fully confluent DLD-1, LoVo, and LS411 cells using confocal microscopy. (b, f, and j) Basal; (c, g, and k) intermediate; and (d, h, and l) apical sections are shown. (e, i, and m) Peptide competition (+ peptide apical). Apical section through DLD-1 (e), LoVo (i), and LS411 cells (m) after preincubation of primary anti–β-catenin and anti-APC antibodies with the neutralizing peptide against which the anti-APC antibody was raised.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196003&req=5

Figure 8: Localization of mutant forms of APC to the apical membrane in colon cancer cell lines. (a) Linear representation of the full-length human APC protein. Several known motifs are shown on top, including the oligomerization domain (oligom.), armadillo repeats (arm. repeats), the 15– and 20–amino acid repeats (both known to bind β-catenin), basic domain, and Dlg binding site. Regions for axin binding are shown below. Immunolocalization of APC (red) and β-catenin (green) in DLD-1 (b–e), LoVo (f–i), and LS411 (j–m) cells. Linear representation of truncated mutant forms of the APC protein expressed by the respective cell line is also shown. Images of successive sections of fully confluent DLD-1, LoVo, and LS411 cells using confocal microscopy. (b, f, and j) Basal; (c, g, and k) intermediate; and (d, h, and l) apical sections are shown. (e, i, and m) Peptide competition (+ peptide apical). Apical section through DLD-1 (e), LoVo (i), and LS411 cells (m) after preincubation of primary anti–β-catenin and anti-APC antibodies with the neutralizing peptide against which the anti-APC antibody was raised.

Mentions: Truncations of APC are loss of function mutations that are responsible for loss of tumor suppressor activity. Therefore, we asked whether truncation mutations in APC affect its apical membrane localization. The distributions of APC in several colon carcinoma cell lines with known truncations of the APC protein were examined by confocal immunofluorescence microscopy. The linear representation of the full-length human APC protein is shown in Fig. 8 a. The DLD-1 cell line (Fig. 8, b–e) harbors a truncation mutation within the central region of the APC protein, a region which is known to be crucial for the tumor suppressor activity of APC through downregulating β-catenin (Munemitsu et al. 1995). The cell line LoVo (Fig. 8, f–i) carries a mutation at codon 1114 of APC, retaining the NH2 terminus of APC including one of the three 15–amino acid repeats. The cell line LS411 (Fig. 8, j–m) carries a mutation at codon 789 which causes chain termination shortly after the seven armadillo repeats (Homfray et al. 1998). A polyclonal antibody directed against the NH2 terminus of the APC protein was used to detect APC. In all cell lines, the mutant APC protein predominantly localized to the apical plasma membrane as observed in serial transverse sections through confluent monolayers. Again, most APC protein did not colocalize with most of the lateral membrane-associated β-catenin in DLD-1, LoVo, and LS411 cells. APC staining was effectively abolished by competition with the specific neutralizing peptide against which the anti-APC antibody was raised (Fig. 8e, Fig. i, and Fig. m). These data suggest that an NH2-terminal fragment of the APC protein containing the oligomerization domain and the arm repeat region is sufficient to localize APC to the apical membrane.


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)

Localization of mutant forms of APC to the apical membrane in colon cancer cell lines. (a) Linear representation of the full-length human APC protein. Several known motifs are shown on top, including the oligomerization domain (oligom.), armadillo repeats (arm. repeats), the 15– and 20–amino acid repeats (both known to bind β-catenin), basic domain, and Dlg binding site. Regions for axin binding are shown below. Immunolocalization of APC (red) and β-catenin (green) in DLD-1 (b–e), LoVo (f–i), and LS411 (j–m) cells. Linear representation of truncated mutant forms of the APC protein expressed by the respective cell line is also shown. Images of successive sections of fully confluent DLD-1, LoVo, and LS411 cells using confocal microscopy. (b, f, and j) Basal; (c, g, and k) intermediate; and (d, h, and l) apical sections are shown. (e, i, and m) Peptide competition (+ peptide apical). Apical section through DLD-1 (e), LoVo (i), and LS411 cells (m) after preincubation of primary anti–β-catenin and anti-APC antibodies with the neutralizing peptide against which the anti-APC antibody was raised.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Localization of mutant forms of APC to the apical membrane in colon cancer cell lines. (a) Linear representation of the full-length human APC protein. Several known motifs are shown on top, including the oligomerization domain (oligom.), armadillo repeats (arm. repeats), the 15– and 20–amino acid repeats (both known to bind β-catenin), basic domain, and Dlg binding site. Regions for axin binding are shown below. Immunolocalization of APC (red) and β-catenin (green) in DLD-1 (b–e), LoVo (f–i), and LS411 (j–m) cells. Linear representation of truncated mutant forms of the APC protein expressed by the respective cell line is also shown. Images of successive sections of fully confluent DLD-1, LoVo, and LS411 cells using confocal microscopy. (b, f, and j) Basal; (c, g, and k) intermediate; and (d, h, and l) apical sections are shown. (e, i, and m) Peptide competition (+ peptide apical). Apical section through DLD-1 (e), LoVo (i), and LS411 cells (m) after preincubation of primary anti–β-catenin and anti-APC antibodies with the neutralizing peptide against which the anti-APC antibody was raised.
Mentions: Truncations of APC are loss of function mutations that are responsible for loss of tumor suppressor activity. Therefore, we asked whether truncation mutations in APC affect its apical membrane localization. The distributions of APC in several colon carcinoma cell lines with known truncations of the APC protein were examined by confocal immunofluorescence microscopy. The linear representation of the full-length human APC protein is shown in Fig. 8 a. The DLD-1 cell line (Fig. 8, b–e) harbors a truncation mutation within the central region of the APC protein, a region which is known to be crucial for the tumor suppressor activity of APC through downregulating β-catenin (Munemitsu et al. 1995). The cell line LoVo (Fig. 8, f–i) carries a mutation at codon 1114 of APC, retaining the NH2 terminus of APC including one of the three 15–amino acid repeats. The cell line LS411 (Fig. 8, j–m) carries a mutation at codon 789 which causes chain termination shortly after the seven armadillo repeats (Homfray et al. 1998). A polyclonal antibody directed against the NH2 terminus of the APC protein was used to detect APC. In all cell lines, the mutant APC protein predominantly localized to the apical plasma membrane as observed in serial transverse sections through confluent monolayers. Again, most APC protein did not colocalize with most of the lateral membrane-associated β-catenin in DLD-1, LoVo, and LS411 cells. APC staining was effectively abolished by competition with the specific neutralizing peptide against which the anti-APC antibody was raised (Fig. 8e, Fig. i, and Fig. m). These data suggest that an NH2-terminal fragment of the APC protein containing the oligomerization domain and the arm repeat region is sufficient to localize APC to the apical membrane.

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