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The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling.

Kunttas-Tatli E, Von Kleeck RA, Greaves BD, Vinson D, Roberts DM, McCartney BM - Mol. Biol. Cell (2015)

Bottom Line: One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin.In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity.Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

No MeSH data available.


Related in: MedlinePlus

Either SAMP repeat is sufficient for the recruitment of APC2 to the destructosome puncta. (A) Schematic representation of mCherry (mCh)-tagged APC2 constructs. Full-length APC2: APC2-FL; APC2 lacking only SAMP1: APC2-S1–/S2+; APC2 lacking only SAMP2: APC2-S1+/S2–; APC2 lacking both SAMP1 and SAMP2: APC2-S1–/S2–. (B) GFP-tagged Axin forms oligomers, which can be visualized as cytoplasmic puncta (1). When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (2), but mCh-APC2-FL expressed without extra Axin is primarily cortical (3). Removal of either of APC2’s SAMP domains (APC2-S1–/S2+ [4] and APC2-S1+/S2– [5]) does not affect colocalization with Axin-GFP, but APC2-S1+/S2– relocalizes Axin to cortex. Removal of both SAMPs (APC2-S1–/S2–) prevents APC2–Axin colocalization (6). Dotted lines indicate cell boundaries. Scale bar, 10 μm.
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Figure 2: Either SAMP repeat is sufficient for the recruitment of APC2 to the destructosome puncta. (A) Schematic representation of mCherry (mCh)-tagged APC2 constructs. Full-length APC2: APC2-FL; APC2 lacking only SAMP1: APC2-S1–/S2+; APC2 lacking only SAMP2: APC2-S1+/S2–; APC2 lacking both SAMP1 and SAMP2: APC2-S1–/S2–. (B) GFP-tagged Axin forms oligomers, which can be visualized as cytoplasmic puncta (1). When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (2), but mCh-APC2-FL expressed without extra Axin is primarily cortical (3). Removal of either of APC2’s SAMP domains (APC2-S1–/S2+ [4] and APC2-S1+/S2– [5]) does not affect colocalization with Axin-GFP, but APC2-S1+/S2– relocalizes Axin to cortex. Removal of both SAMPs (APC2-S1–/S2–) prevents APC2–Axin colocalization (6). Dotted lines indicate cell boundaries. Scale bar, 10 μm.

Mentions: Overexpressed or endogenous Axin in cell culture and intact tissues forms cytoplasmic puncta due in part to its ability to form oligomers (Fagotto et al., 1999; Faux et al., 2008; Fiedler et al., 2011). In Drosophila cultured S2 cells, APC2 is recruited into these puncta via its SAMP repeats (Kunttas-Tatli et al., 2014; and compare Figure 2B, 2 to 6), although APC and Axin can interact via SAMP-independent mechanisms as well (Roberts et al., 2011; Schneikert et al., 2014). We used this system to ask whether the differences in Axin binding we observed by Y2H affected APC2’s ability to be recruited to the destructosome by Axin. To determine whether APC2 SAMP1 or SAMP2 has a more significant role in destructosome localization, we generated mutants that lacked SAMP1 (APC2-S1–/S2+), SAMP2 (APC2-S1+/S2–), or both (APC2-S1–/S2–; Figure 2A). While generating these deletion mutants, we retained the last 30 amino acids (C30) because this sequence is required for the cortical localization of Drosophila APC2 (Zhou et al., 2011). We coexpressed the APC2 SAMP mutants (APC2-S1–/S2+ and APC2-S1+/S2–) with Axin–green fluorescent protein (GFP; Figure 2B, 4 and 5). Whereas both SAMPs have the ability to bind Axin by Y2H (Figure 1C), we predicted that the weaker binder, SAMP1, would have reduced ability to facilitate recruitment. However, we observed that either SAMP repeat was sufficient for destructosome recruitment. Surprisingly, expression of APC2-S1+/S2– (lacking the stronger Axin binder, SAMP2) resulted in redistribution of Axin to the cell cortex in a proportion of the cells (Supplemental Figure S2A, 1–3). Because the ability of Axin to form puncta might depend on its expression level, we used fluorescence-activated cell sorting (FACS) to sort the cells on the basis of their Axin expression levels. This revealed that Axin redistribution to the cortex in the presence of APC2 was enhanced in cells expressing high levels of Axin-GFP (Supplemental Figure S2B).


The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling.

Kunttas-Tatli E, Von Kleeck RA, Greaves BD, Vinson D, Roberts DM, McCartney BM - Mol. Biol. Cell (2015)

Either SAMP repeat is sufficient for the recruitment of APC2 to the destructosome puncta. (A) Schematic representation of mCherry (mCh)-tagged APC2 constructs. Full-length APC2: APC2-FL; APC2 lacking only SAMP1: APC2-S1–/S2+; APC2 lacking only SAMP2: APC2-S1+/S2–; APC2 lacking both SAMP1 and SAMP2: APC2-S1–/S2–. (B) GFP-tagged Axin forms oligomers, which can be visualized as cytoplasmic puncta (1). When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (2), but mCh-APC2-FL expressed without extra Axin is primarily cortical (3). Removal of either of APC2’s SAMP domains (APC2-S1–/S2+ [4] and APC2-S1+/S2– [5]) does not affect colocalization with Axin-GFP, but APC2-S1+/S2– relocalizes Axin to cortex. Removal of both SAMPs (APC2-S1–/S2–) prevents APC2–Axin colocalization (6). Dotted lines indicate cell boundaries. Scale bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Either SAMP repeat is sufficient for the recruitment of APC2 to the destructosome puncta. (A) Schematic representation of mCherry (mCh)-tagged APC2 constructs. Full-length APC2: APC2-FL; APC2 lacking only SAMP1: APC2-S1–/S2+; APC2 lacking only SAMP2: APC2-S1+/S2–; APC2 lacking both SAMP1 and SAMP2: APC2-S1–/S2–. (B) GFP-tagged Axin forms oligomers, which can be visualized as cytoplasmic puncta (1). When coexpressed, mCh-APC2-FL colocalizes in cytoplasmic puncta with Axin-GFP (2), but mCh-APC2-FL expressed without extra Axin is primarily cortical (3). Removal of either of APC2’s SAMP domains (APC2-S1–/S2+ [4] and APC2-S1+/S2– [5]) does not affect colocalization with Axin-GFP, but APC2-S1+/S2– relocalizes Axin to cortex. Removal of both SAMPs (APC2-S1–/S2–) prevents APC2–Axin colocalization (6). Dotted lines indicate cell boundaries. Scale bar, 10 μm.
Mentions: Overexpressed or endogenous Axin in cell culture and intact tissues forms cytoplasmic puncta due in part to its ability to form oligomers (Fagotto et al., 1999; Faux et al., 2008; Fiedler et al., 2011). In Drosophila cultured S2 cells, APC2 is recruited into these puncta via its SAMP repeats (Kunttas-Tatli et al., 2014; and compare Figure 2B, 2 to 6), although APC and Axin can interact via SAMP-independent mechanisms as well (Roberts et al., 2011; Schneikert et al., 2014). We used this system to ask whether the differences in Axin binding we observed by Y2H affected APC2’s ability to be recruited to the destructosome by Axin. To determine whether APC2 SAMP1 or SAMP2 has a more significant role in destructosome localization, we generated mutants that lacked SAMP1 (APC2-S1–/S2+), SAMP2 (APC2-S1+/S2–), or both (APC2-S1–/S2–; Figure 2A). While generating these deletion mutants, we retained the last 30 amino acids (C30) because this sequence is required for the cortical localization of Drosophila APC2 (Zhou et al., 2011). We coexpressed the APC2 SAMP mutants (APC2-S1–/S2+ and APC2-S1+/S2–) with Axin–green fluorescent protein (GFP; Figure 2B, 4 and 5). Whereas both SAMPs have the ability to bind Axin by Y2H (Figure 1C), we predicted that the weaker binder, SAMP1, would have reduced ability to facilitate recruitment. However, we observed that either SAMP repeat was sufficient for destructosome recruitment. Surprisingly, expression of APC2-S1+/S2– (lacking the stronger Axin binder, SAMP2) resulted in redistribution of Axin to the cell cortex in a proportion of the cells (Supplemental Figure S2A, 1–3). Because the ability of Axin to form puncta might depend on its expression level, we used fluorescence-activated cell sorting (FACS) to sort the cells on the basis of their Axin expression levels. This revealed that Axin redistribution to the cortex in the presence of APC2 was enhanced in cells expressing high levels of Axin-GFP (Supplemental Figure S2B).

Bottom Line: One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin.In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity.Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.

No MeSH data available.


Related in: MedlinePlus