A few key signal transduction pathways shape cell fate decisions during normal development and maintain adult tissue homeostasis. These powerful pathways must be kept under tight control, as each also plays critical roles in oncogenesis, with virtually every tumor exhibiting inappropriate activation of one or more pathways. One of the most interesting surprises in signal transduction has been the discovery of elaborate machinery that evolved to keep these pathways tightly off in the absence of ligands. This negative regulatory machinery is the target of inactivating mutations in human tumors.
The Wnt pathway provides a striking example (Cadigan and Peifer, 2009
; Chien et al., 2009
). It is negatively regulated at many levels, from secreted Wnt antagonists to repressors binding T-cell factor (TCF) transcription factors at the end of the pathway. However, the primary mechanism keeping signaling off in cells not receiving Wnt ligands is regulated destruction of the key effector β-catenin (βcat; fly homologue Armadillo [Arm]). βcat is constitutively phosphorylated by a set of proteins known as the destruction complex, which triggers ubiquitination and proteasomal destruction. Wnt signals inactivate the destruction complex, stabilizing βcat and allowing it to enter nuclei to act as a transcriptional coactivator. In colon and other cancers, constitutive activation of Wnt signaling, by gain-of-function βcat mutations preventing its destruction or loss-of-function mutations in the destruction complex proteins adenomatous polyposis coli (APC) or Axin, plays an important role (Polakis, 2007
mutations occur in >80% of all colon cancers, and thus APC's mechanistic roles in Wnt signaling are of significant interest.
APC is a multidomain protein regulating both Wnt signaling and the cytoskeleton (McCartney and Nathke, 2008
; Bahmanyar et al., 2009
; ). N-terminal in APC family proteins is an Arm-repeat domain (so-called because it was first found in βcat/Arm), a protein interaction domain known to bind several partners, most with cytoskeletal functions, and also critical for regulating Wnt signaling, perhaps through an as yet unidentified protein partner. APC's middle region carries multiple binding sites for its destruction complex partner Axin (the Ser-Ala-Met-Pro [SAMP] repeats) and for βcat (the 15- and 20-aa repeats). C-terminal in mammalian APC is a region interacting directly and indirectly with microtubules (MTs). However, this latter region, while likely important for cytoskeletal regulation, is not essential for Wnt regulation, as mouse mutants lacking this region are adult-viable and not tumor-prone (Smits et al., 1999
). Further, fly APC2, which can regulate Wnt signaling, ends after the SAMP motifs, thus lacking the direct MT-interacting domains ().
FIGURE 1: Diagrams of wild-type APC2 and the mutants used in both the localization studies and the functional analyses, and a summary of the functions of each mutant. Scale is in amino acids. (A) Both flies and mammals have two APC family members that share a core, (more ...)
Despite 15 yr of work, key questions remain about APC's mechanistic role in Wnt regulation. APC is essential for effectively targeting βcat for phosphorylation and destruction, but its role in the destruction complex is not clear. It was initially assumed to be the scaffold for presenting βcat to the kinases CK1 and GSK3 (Rubinfeld et al., 1996
), but now it is clear that Axin plays this role (Ha et al., 2004
). Another model, which we have favored (McCartney et al., 2006
), suggests that APC binds the destruction complex and, via other protein interactions, localizes it to the correct place in the cell. However, it remains unclear where within the cell the destruction complex normally operates.
Mammalian APC has a complex subcellular localization mediated, at least in part, by cytoskeletal interactions. In isolated cultured cells it localizes to the cortex, particularly in cell protrusions, at which it clusters at ends of MTs (Näthke et al., 1996
). It also can be transported along MTs and associate with MT plus ends (Mimori-Kiyosue et al., 2000
). In epithelial cells and tissues, APC localization is more controversial, but most studies suggest at least a pool localizes to cell–cell junctions or the basolateral cell cortex (e.g., Langford et al., 2006a
; Grohmann et al., 2007
; Hendriksen et al., 2008
; Maher et al., 2009
). Fly APC2 localizes to the cell cortex (McCartney et al., 1999
; Yu et al., 1999
). The Arm repeats and C-terminal end of APC2 (McCartney et al., 2006
; Zhou et al., 2011
) are important for this cortical localization, but the mechanisms of cortical localization of APC family proteins remain mysterious.
This led to the hypothesis that APC localizes the destruction complex to the cell cortex, thus facilitating its function. A cortical location would put it in proximity to the Wnt receptors (Hendriksen et al., 2008
), allowing rapid down-modulation after Wnt signaling. Both fly APC2 (McCartney et al., 1999
) and human APC (Näthke et al., 1996
) accumulate, at least in part, at the cell cortex. Consistent with the idea that cortical localization is essential for Wnt regulation, missense APC2
alleles exhibit a strong correlation between loss of cortical localization and loss of function in Wnt regulation (McCartney et al., 2006
), and one, APC2S
, is temperature-sensitive in phenotype and
localization to the cell cortex (McCartney et al., 1999
). However, other data mitigate against this model. While the two fly APC family members, APC1 and APC2, are redundant for Wnt regulation in many tissues (Ahmed et al., 2002
; Akong et al., 2002a
), their predominant intracellular localizations are distinct. APC2 is cortical, but APC1 primarily localizes to axons in neurons and to centrosomes and associated MTs in male germ line stem cells (Yamashita et al., 2003
) or when overexpressed in the ectoderm or neuroblasts (Akong et al., 2002a
). This calls the localization model into question. However, each APC can recruit the other to its “favorite location” when overexpressed (Akong et al., 2002a
), and APC1 and APC2 interact in a two-hybrid assay (Mattie et al., 2010
), raising the possibility that APC1 is recruited to the cortex at a low, but still functional, level. In this work, we test the localization model directly by altering APC2 localization and evaluating effects on its function.
In addition to suggesting a role for APC in the destruction complex, most reviews of Wnt signaling propose that APC also acts in nuclei in βcat regulation (Brocardo and Henderson, 2008
; Neufeld, 2009
). One model suggests that APC shuttles in and out of nuclei, exporting βcat from the nucleus to inactivate it (Bienz, 2002
). Consistent with this, APC proteins have nuclear localization (NLS) and nuclear export signals (NES) and accumulate in nuclei after nuclear export is blocked by leptomycin B treatment (Henderson, 2000
; Neufeld et al., 2000
; Rosin-Arbesfeld et al., 2003
). APC also can physically associate with Wnt target genes (Sierra et al., 2006
), raising the possibility that it plays a direct role in repressing Wnt target genes. Finally, APC can bind the transcriptional repressor C-terminal binding protein (CtBP; Hamada and Bienz, 2004
; Schneikert et al., 2011
). While most functional tests of these nuclear roles have been indirect, involving misexpression of truncated proteins rather than genetic tests in vivo, Tolwinski (2009
) found that membrane-tethered, myristylated APC2, which should be prevented from entering nuclei, cannot rescue Wnt regulation, consistent with an essential nuclear role. However, that study only used a single membrane-tethered version of APC2, and it is possible that the N-terminal tag inactivated protein function, leaving open the possibility that nuclear localization is not essential.
We thus set out to resolve whether APC plays a key role in targeting the destruction complex to a critical subcellular position, and whether APC needs to enter the nucleus to regulate Wnt signaling. We tested these hypotheses in both cultured colon cancer cells and the fruit fly Drosophila, allowing us to determine whether any such roles are conserved or might be divergent between flies and mammals.