Cortical localization of APC2 plays a role in actin organization but not in Wnt signaling in<i>Drosophila</i>

Zhou, Meng-Ning; Kunttas-Tatli, Ezgi; Zimmerman, Sandra G; Zhouzheng, Fangyuan; McCartney, Brooke M.

doi:10.1242/jcs.073916

Cited by 22 publications

(58 citation statements)

References 58 publications

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“…To begin the characterization of these mutant proteins, we expressed them in Drosophila S2 cells. We have previously shown that APC2-FL localizes to the cortex in S2 cells, similar to its endogenous localization in embryonic cells (Zhou et al 2011). This localization requires both the Arm repeats and the C-terminal C30 domain (Zhou et al 2011), all of which are intact in our mutants.…”

Section: Resultssupporting

confidence: 76%

“…We have previously shown that APC2-FL localizes to the cortex in S2 cells, similar to its endogenous localization in embryonic cells (Zhou et al 2011). This localization requires both the Arm repeats and the C-terminal C30 domain (Zhou et al 2011), all of which are intact in our mutants. As a test of the stability and functionality of our mutants, we asked whether they localized to the S2 cell cortex.…”

Section: Resultssupporting

confidence: 76%

“…Expression was induced at 24 hr post-transfection by adding CuSO 4 to a final concentration of 40 mM for 14-16 hr. Co-immunoprecipitation (co-IP) was performed as in Zhou et al (2011). In brief, after induction cells were lysed and the lysate was preincubated with rec-G beads (Invitrogen) for 0.5 hr at 4°.…”

Section: Image Analysismentioning

confidence: 99%

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Destruction Complex Function in the Wnt Signaling Pathway ofDrosophilaRequires Multiple Interactions Between Adenomatous Polyposis Coli 2 and Armadillo

et al. 2012

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The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, b-catenin (bcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to bcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds bcat and the affinity is significantly increased. These distinct APC-bcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for bcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.

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Section: Resultssupporting

confidence: 76%

Section: Resultssupporting

confidence: 76%

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Destruction Complex Function in the Wnt Signaling Pathway ofDrosophilaRequires Multiple Interactions Between Adenomatous Polyposis Coli 2 and Armadillo

et al. 2012

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“…A pool of APC localizes to the basolateral cortex or cell-cell junctions of vertebrate epithelial cells and tissues, and Drosophila APC2 likewise localizes to the cell cortex through interactions with the microtubule and actin cytoskeletons (Näthke et al 1996;McCartney et al 1999McCartney et al , 2006Yu et al 1999;Rosin-Arbesfeld et al 2001;Langford et al 2006a,b;Grohmann et al 2007;Zhou et al 2011). In studies of nuclear export by APC, it was found that truncated APCs lacking cytoskeletal interaction regions shuttle between the nucleus and cytoplasm more efficiently than the full-length protein, suggesting that cytoskeletal interactions help to retain APC in the cytosol (Brocardo et al 2005), consistent with a role in cytoplasmic retention of b-catenin.…”

Section: Destruction Complex Localizationmentioning

confidence: 99%

The -Catenin Destruction Complex

Stamos

Weis

2012

Cold Spring Harbor Perspectives in Biology

893

783

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The Wnt/b-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator b-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase b-TrCP. The complex generates a b-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the b-catenin amino terminus, a process that requires scaffolding of the kinases and bcatenin by Axin. Ubiquitinated b-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in b-catenin destruction.

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“…APC proteins are thought to dimerize/ oligomerize as vertebrate APC proteins contain an N-terminal coiled-coil (Joslyn et al 1993) and Drosophila APC2 has been shown to self-associate in immunoprecipitation studies (Zhou et al 2011;Roberts et al 2012a). In addition, a yeast twohybrid study suggests that Drosophila APC1 and APC2 proteins can heterodimerize (Mattie et al 2010).…”

Section: An Apc2 Mutant Lacking All Bcat-binding Sites Is Also Functimentioning

confidence: 99%

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

Yamulla¹,

Kane²,

Moody³

et al. 2014

Genetics

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The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in .80% of colorectal cancers. APC participates in a multiprotein "destruction complex" that targets the proto-oncogene b-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity b-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all b-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that b-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all b-catenin-binding sites (including the 15Rs) and find that a direct b-catenin/APC interaction is also not essential for b-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby b-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock b-catenin in the destruction complex to increase the efficiency of b-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of b-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of b-catenin likely accounts for this difference.

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Cortical localization of APC2 plays a role in actin organization but not in Wnt signaling inDrosophila

Cited by 22 publications

References 58 publications

Destruction Complex Function in the Wnt Signaling Pathway ofDrosophilaRequires Multiple Interactions Between Adenomatous Polyposis Coli 2 and Armadillo

Destruction Complex Function in the Wnt Signaling Pathway ofDrosophilaRequires Multiple Interactions Between Adenomatous Polyposis Coli 2 and Armadillo

The -Catenin Destruction Complex

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

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