APC: the plot thickens.

Adenomatous polyposis coli (APC) is an important tumour suppressor in the human colon. It is conserved between human and flies, and promotes, together with Axin and glycogen synthase kinase 3 (GSK3), the degradation of the Wnt-signalling effector beta-catenin. Recent experiments have shaped our understanding of how Axin and GSK3 function but the role of APC in this process remains elusive.     

Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function

The tumor suppressor adenomatous polyposis coli (APC) plays a critical role in the turnover of cytosolic beta-catenin, the key effector of the canonical Wnt signaling pathway. APC contains seven 20 amino acid (20 aa) beta-catenin binding repeats that are required for beta-catenin turnover. We have determined the crystal structure of beta-catenin in complex with a phosphorylated APC fragment containing two 20 aa repeats. Surprisingly, one single phosphorylated 20 aa repeat, together with its flanking regions, covers the entire structural groove of beta-catenin and may thus compete for beta-catenin binding with all other beta-catenin armadillo repeat partners. Our biochemical studies show that phosphorylation of the APC 20 aa repeats increases the affinity of the repeats for beta-catenin by 300- to 500-fold and the phosphorylated 20 aa repeats prevent beta-catenin binding to Tcf. Our work suggests that the phosphorylation of the APC 20 aa repeats could be a critical switch for APC function.     

Human Kid is Degraded by the APC/CCdh1 but Not by the APC/CCdc20

The APC/CCdh1 (Anaphase Promoting Complex/Cyclosome) targets numerous cell cycle proteins for ubiquitin mediated degradation in late mitosis and G1. The KEN box is one of two major recognition motifs of APC/CCdh1 substrates. This motif is however very common and shared by a tenth of the human proteome, the vast majority of which are obviously not APC/C substrates. We have observed that most known functional KEN boxes are followed by a proline residue and show that this proline plays a role in APC/CCdh1 specific degradation. This insight can be instrumental for identifying novel APC/CCdh1 substrates. We used this KENxP motif to identify human Aurora B and Kid as APC/CCdh1 substrates. The degradation of Xenopus XKid at metaphase by APC/CCdc20 is essential for chromatid segregation. Human Kid in contrast is degraded later and its APC/CCdh1 specific degradation is not required for mitotic progress. It is thus likely that Kid inactivation in G1 takes place both by nuclear sequestration and degradation by the APC/CCdh1.     

APC2 Cullin Protein and APC11 RING Protein Comprise the Minimal Ubiquitin Ligase Module of the Anaphase-promoting Complex

In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.     

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.     

Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. We further explored the functions of the Drosophila APCs using the larval brain as a model. We found that both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. Our experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins.     

Macrophages as APC and the dendritic cell myth

Dendritic cells have been considered an immune cell type that is specialized for the presentation of Ag to naive T cells. Considerable effort has been applied to separate their lineage, pathways of differentiation, and effectiveness in Ag presentation from those of macrophages. This review summarizes evidence that dendritic cells are a part of the mononuclear phagocyte system and are derived from a common precursor, responsive to the same growth factors (including CSF-1), express the same surface markers (including CD11c), and have no unique adaptation for Ag presentation that is not shared by other macrophages.     

Regulation of the APC and the exit from mitosis

The events of late mitosis, from sister-chromatid separation to cytokinesis, are governed by the anaphase-promoting complex (APC), a multisubunit assembly that triggers the ubiquitin-dependent proteloysis of key regulatory proteins. An intricate regulatory network governs APC activity and helps to ensure that late mitotic events are properly timed and coordinated.     

Expansion of the APC superfamily of secondary carriers

The amino acid‐polyamine‐organoCation (APC) superfamily is the second largest superfamily of secondary carriers currently known. In this study, we establish homology between previously recognized APC superfamily members and proteins of seven new families. These families include the PAAP (Putative Amino Acid Permease), LIVCS (Branched Chain Amino Acid:Cation Symporter), NRAMP (Natural Resistance‐Associated Macrophage Protein), CstA (Carbon starvation A protein), KUP (K⁺ Uptake Permease), BenE (Benzoate:H⁺ Virginia Symporter), and AE (Anion Exchanger). The topology of the well‐characterized human Anion Exchanger 1 (AE1) conforms to a UraA‐like topology of 14 TMSs (12 α‐helical TMSs and 2 mixed coil/helical TMSs). All functionally characterized members of the APC superfamily use cation symport for substrate accumulation except for some members of the AE family which frequently use anion:anion exchange. We show how the different topologies fit into the framework of the common LeuT‐like fold, defined earlier (Proteins. 2014 Feb;82(2):336‐46), and determine that some of the new members contain previously undocumented topological variations. All new entries contain the two 5 or 7 TMS APC superfamily repeat units, sometimes with extra TMSs at the ends, the variations being greatest within the CstA family. New, functionally characterized members transport amino acids, peptides, and inorganic anions or cations. Except for anions, these are typical substrates of established APC superfamily members. Active site TMSs are rich in glycyl residues in variable but conserved constellations. This work expands the APC superfamily and our understanding of its topological variations. Proteins 2014; 82:2797–2811. © 2014 Wiley Periodicals, Inc.     

Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract

Purpose

APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumour risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution creates an oligo-adenine tract (A8) that appears to be inherently prone to further mis-pairing and slippage. The reported multiple tumor phenotype of carriers is not easily reconciled with molecular and population genetics data. We postulated that some c.3920T>A carriers with multiple adenomas have other unidentified APC germ line or somatic mutations.

Methods

DNA from 82 colonic tumours and accompanying normal tissue collected from 29 carriers with multiple colorectal tumors was directly sequenced between codons 716 and 1604. We also assessed APC gene loss of heterozygosity.

Results

One patient (3.4%) was found to have an additional APC germ line mutation. Twenty-five of the tumours showed no significant somatic molecular change, 36 showed one change, 20 showed two, and one tumour showed more than 2 changes. Our data suggest a correlation between advancing histology and fewer beta-catenin binding sites remaining in the mutant proteins.

Conclusions

There were no other common germ line variants identified within the region of the APC gene examined, suggesting that any effect from this region on tumour production is attributable to the c.3920T>A allele. Our findings further suggest the only somatic genetic change clearly attributable to the c.3920T>A mutation is the c.3924_3925insA.