Domain Of Adenomatous Polyposis Coli Research Articles

In silico evaluation of anti-colorectal cancer inhibitors by Resveratrol derivatives targeting Armadillo repeats domain of APC: molecular docking and molecular dynamics simulation.

Colorectal cancer is the second leading cause of cancer-related deaths. In 2018, there were an estimated 1.8 million cases, and this number is expected to increase to 2.2 million by 2030. Despite its prevalence, the current therapeutic option has a lot of side effects and limitations. Therefore, this study was designed to employ a computational approach for the identification of anti-cancer inhibitors against colorectal cancer using Resveratrol derivatives. Initially, the pass prediction spectrum of 50 derivatives was conducted and selected top seven compounds based on the maximum pass prediction score. After that, a comprehensive analysis, including Lipinski Rule, pharmacokinetics, ADMET profile study, molecular orbitals analysis, molecular docking, molecular dynamic simulations, and MM-PBSA binding free energy calculations. The reported binding affinity ranges of Resveratrol derivatives from molecular docking were -6.1 kcal/mol to -7.9 kcal/mol against the targeted receptor of human armadillo repeats domain of adenomatous polyposis coli (APC) (PDB ID: 3NMW). Specifically, our findings reported that two compounds [(03) Resveratrol 3-beta-mono-D-glucoside, and (29) Resveratrol 3-Glucoside] displayed the highest level of effectiveness compared to all other derivatives (-7.7 kcal/mol and -7.9 kcal/mol), and favorable drug-likeness, and exceptional safety profiles. Importantly, almost all the molecules were reported as free from toxic effects. Subsequently, molecular dynamic simulations conducted over 100ns confirmed the stability of the top two ligand-protein complexes. These findings suggest that Resveratrol derivatives may be effective drug candidate to manage the colorectal cancer. However, further experimental research, such as in vitro/in vivo studies, is essential to validate these computational findings and confirm their practical value.

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ‘ACTIF’ that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.

EB1 Directly Regulates APC-Mediated Actin Nucleation

EB1 was discovered 25 years ago as a binding partner of the tumor suppressor adenomatous polyposis coli (APC) [1]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [2-5], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [6, 7]. Whether EB1 interactions govern functions of APC beyond microtubule regulation has not been explored. The C-terminal basic domain of APC (APC-B) directly nucleates actin assembly, and this activity is required invivo for directed cell migration and for maintaining normal levels of F-actin [8-10]. Here, we show that EB1 binds APC-B and inhibits its actin nucleation function by blocking actin monomer recruitment. Consistent with these biochemical observations, knocking down EB1 increases F-actin levels in cells, and this can be rescued by disrupting APC-mediated actin nucleation. Conversely, overexpressing EB1 decreases F-actin levels and impairs directed cell migration without altering microtubule organization and independent of its direct binding interactions with microtubules. Overall, our results define a new function for EB1 in negatively regulating APC-mediated actin assembly. Combining these findings with other recent studies showing that APC interactions regulate EB1-dependent effects on microtubule dynamics [7], we propose that EB1-APC interactions govern bidirectional cytoskeletal crosstalk by coordinating microtubule and actin dynamics.

Adenomatous Polyposis Coli as a Scaffold for Microtubule End-Binding Proteins

End-binding proteins (EBs), referred to as the core components of the microtubule plus-end tracking protein network, interact with the C-terminus of the adenomatous polyposis coli (APC) tumor suppressor. This interaction is disrupted in colon cancers expressing truncated APC. APC and EBs act in synergy to regulate microtubule dynamics during spindle formation, chromosome segregation and cell migration. Since EBs autonomously end-track microtubules and partially co-localize with APC at microtubule tips in cells, EBs have been proposed to direct APC to microtubule ends. However, the interdependency of EB and APC localization on microtubules remains elusive. Here, using in vitro reconstitution and single-molecule imaging, we have investigated the interplay between EBs and the C-terminal domain of APC (APC-C) on dynamic microtubules. Our results show that APC-C binds along the microtubule wall but does not accumulate at microtubule tips, even when EB proteins are present. APC-C was also found to enhance EB binding at the extremity of growing microtubules and on the microtubule lattice: APC-C promotes EB end-tracking properties by increasing the time EBs spend at microtubule growing ends, whereas a pool of EBs with a fast turnover accumulates along the microtubule surface. Overall, our results suggest that APC is a promoter of EB interaction with microtubules, providing molecular determinants to reassess the relationship between APC and EBs.

N-terminal and central domains of APC function to regulate branch number, length and angle in developing optic axonal arbors in vivo

During formation of neuronal circuits, axons navigate long distances to reach their target locations in the brain. When axons arrive at their target tissues, in many cases, they extend collateral branches and/or terminal arbors that serve to increase the number of synaptic connections they make with target neurons. Here, we investigated how Adenomatous Polyposis Coli (APC) regulates terminal arborization of optic axons in living Xenopus laevis tadpoles. The N-terminal and central domains of APC that regulate the microtubule cytoskeleton and stability of β-catenin in the Wnt pathway, were co-expressed with GFP in individual optic axons, and their terminal arbors were then imaged in tectal midbrains of intact tadpoles. Our data show that the APCNTERM and APCβ-cat domains both decreased the mean number, and increased the mean length, of branches in optic axonal arbors relative to control arbors in vivo. Additional analysis demonstrated that expression of the APCNTERM domain increased the average bifurcation angle of branching in optic axonal arbors. However, the APCβ-cat domain did not significantly affect the mean branch angle of arbors in tecta of living tadpoles. These data suggest that APC N-terminal and central domains both modulate number and mean length of branches optic axonal arbors in a compensatory manner, but also define a specific function for the N-terminal domain of APC in regulating branch angle in optic axonal arbors in vivo. Our findings establish novel mechanisms for the multifunctional protein APC in shaping terminal arbors in the visual circuit of the developing vertebrate brain.

A disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

The adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of APC, we explored APC 1638T/1638T (APC1638T) mice that express a truncated APC lacking the C-terminal domain. The APC1638T mice were tumor free and exhibited growth retardation. In the present study, we compared small intestinal crypt-villus cells homeostasis in APC +/+ (WT) mice and APC1638T mice. The body weight of APC1638T mice was significantly smaller than that of WT mice at all ages. The length of small intestine of APC1638T mice was significantly shorter than that of WT mice. The crypt-villus axis was significantly elongated, and the number of intestinal epithelial cells also increased in APC1638T mice compared with those in WT mice. However, the number of intestinal epithelial cells per 100µm of villi was not different between WT and APC1638T mice. Migration and proliferation of intestinal epithelial cells in APC1638T mice were faster than that in WT mice. The population of Goblet cells, Paneth cells, and enteroendocrine cells was significantly altered in APC1638T mice. These results indicate that C-terminal domain of APC has a role in the regulation of intestinal epithelium homeostasis.

Abstract 3671: Polarity and tumor development: Regulation by adenomatous polyposis coli

Abstract The process of epithelial polarity is tightly regulated by multiple complexes including aPKC, PAR, and Crumbs proteins. Our laboratory has recently investigated loss of Adenomatous Polyposis Coli (APC) in the MDCK 3D culture system. We have observed a lack of hollow lumens and mislocalization of podocalyxin (gp135) to the basal surface, both of which are independent of the canonical Wnt/β-catenin signaling pathway. We further showed that the c-terminus of APC, which contains binding domains for polarity proteins (Dlg and Scrib) and cytoskeletal proteins (microtubules and actin), is responsible for the observed phenotypes. In the current study, we have investigated the molecular mechanisms required for the inversion of polarity and the potential impact on tumorigenesis. We have made the novel observation that different mechanisms are responsible for the increased size versus altered polarity observed in cysts derived from APC-KD cells. Alterations in size, although independent of changes in proliferation and/or apoptosis, are regulated by the c-terminal domain, focal adhesion kinase (FAK), Src, and β1-integrin. Despite this, the mis-localization of gp135 is only rescued by the c-terminal domain of APC, suggesting a unique molecular mechanism. As loss of epithelial polarity is an early marker for tumor development, we investigated tumor-associated phenotypes. APC-KD cells were found to grow larger colonies in soft agar, and have increased migration in wound-filling assays, which appeared to be regulated through similar molecular mechanisms. These data suggest that APC regulates polarity and associated disease phenotypes (including kidney development, cardiovascular disease, and cancer) through pathways independent of Wnt signaling. Citation Format: Alyssa C. Lesko, Madeline Chandra, Jenifer R. Prosperi. Polarity and tumor development: Regulation by adenomatous polyposis coli. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3671.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.

Abstract 1986: The role of APC in chemotherapeutic responsiveness of breast cancer

Abstract Breast cancer is the leading cause of cancer-related death in non-smoking women. Despite tremendous amounts of information about the etiology of breast cancer, many questions remain unanswered. It has been demonstrated that specific subtypes of breast cancer respond differently to chemotherapeutic and targeted therapies. The oncogenic events and signaling pathways driving these tumor subtypes are distinct, indicating that increased knowledge of their molecular basis will provide opportunities for novel therapeutic approaches and knowledge of therapeutic responsiveness, to ultimately improve patient outcomes. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in 18-70% of sporadic breast cancers depending on subtype; however, the effect of APC mutation on tumorigenic properties remains unexplored. APC binds both directly and indirectly to microtubules and regulates multiple components of the DNA repair pathway, suggesting it may play a critical role in therapeutic responsiveness. Using the ApcMin/+ mouse model, we previously identified pre-neoplastic lesions in the breast, alterations in genes critical in therapeutic resistance, and enhanced breast tumorigenesis in the presence of the Polyoma middle T antigen (PyMT) oncogene independent of Wnt/β-catenin signaling. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. Cell lines derived from PyMT-mediated tumors demonstrated alterations in expression of two ATP-binding cassette transporters, multidrug resistance protein 1 (MDR1) and ATP-binding cassette sub-family G member 2/Breast Cancer Resistance Protein (ABCG2/BCRP), both of which are critical in predicting responsiveness to therapeutic agents. Given that one aspect of therapeutic resistance is an increase in tumor initiating cells (TIC), an ALDEFLUOR assay was performed and surprisingly showed a decrease in TIC population in cells from MMTV-PyMT;ApcMin/+ mice. In addition, cells from MMTV-PyMT;ApcMin/+ mice were more sensitive to paclitaxel and 5-fluorouracil via MTS assay. To translate our findings to a human breast cancer cell line, we utilized DU4475 cells, which harbor a mutation in APC in the β-catenin binding region. We have generated stable DU4475 cell lines expressing the middle region or C-terminal domain of APC, and showed that these cell lines have increased sensitivity to paclitaxel, 5-fluorouracil, cisplatin, and doxorubicin. Combined, these data suggest that APC does modulate therapeutic resistance in breast cancer cells in a context-dependent manner. Future studies will involve the continued studies in the DU4475 cohort of cells and testing the specificity of APC in regulating the therapeutic response in the PyMT-derived cell lines. Citation Format: Katia Fernandez Soto, Monica K. VanKlompenberg, Jenifer R. Prosperi. The role of APC in chemotherapeutic responsiveness of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1986. doi:10.1158/1538-7445.AM2014-1986

Abstract P5-07-08: Restoration of APC decreases tumorigenic potential in breast cancer cells

Abstract Mutations of the Adenomatous Polyposis Coli (APC) tumor suppressor gene have been reported in 5-70% of human breast cancers. APC is most well known for its role as a negative regulator of the Wnt/b-catenin pathway, where APC sequesters β-catenin and targets it for degradation via the proteasome. The breast cancer cell line, DU4475, has a single point mutation in the mutation cluster region of APC, coding for an early stop and subsequent truncated protein, lacking the β-catenin binding domain. Preliminary data using a TOPFlash reporter assay suggested that Wnt/β-catenin pathway was active, which was validated with immunofluorescence (IF) showing nuclear localization of β-catenin. Our overall hypothesis is that restoration of APC will decrease tumorigenic potential in DU4475 breast cancer cells. To test our hypothesis, we have generated DU4475 cells stably expressing GFP (control) (DU4475-GFP), a GFP-tagged internal fragment of APC that includes the β-catenin binding domain (DU4475-APCmid), and a GFP-tagged C-terminal fragment of APC that includes the microtubule binding domain of APC (DU4475-APCC-ter). After selection using G418, the GFP positive cells were sorted using a cell sorter. Results from TOPFlash reporter assay and IF demonstrated a modest decrease in Wnt/β-catenin activity in DU4475-APCmid cells. In addition, we observed decreased proliferation as measured in a growth assay in DU4475-APCmid cells compared to the control cells. We also observed a 17% increase in cleaved caspase-3 and only a minimal decrease in cell proliferation as measured by phospho-Histone H3 using IF in 3D cultures upon APCmid re-introduction. We also observed decreases in Cyclin D1, Cyclin B1, Early Growth Response 1, and HIF1α in DU4475-APCmid using real time PCR. These changes suggest that the re-introduction of the central domain of the APC tumor suppressor results in alterations in cell growth and gene expression changes indicative of an increased tumorigenic phenotype in vitro. Ongoing studies in the laboratory are dissecting the signaling pathways downstream of APC restoration to identify therapeutic targets that may be used to restore the functional alterations of mutant APC. These studies have generated a system to be utilized for in vivo tumor and metastasis formation under the control of APC mutation in a human cell culture model. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-08.

Adenomatous polyposis coli (APC) membrane recruitment 3, a member of the APC membrane recruitment family of APC‐binding proteins, is a positive regulator of Wnt–β‐catenin signalling

The adenomatous polyposis coli (APC) membrane recruitment (Amer) family proteins Amer1/Wilms tumour gene on the X chromosome and Amer2 are binding partners of the APC tumour suppressor protein, and act as negative regulators in the Wnt signalling cascade. So far, nothing has been known about the third member of the family, Amer3. Here we show that Amer3 binds to the armadillo repeat domain of APC, similarly to Amer1 and Amer2. Amer3 also binds to the Wnt pathway regulator conductin/axin2. Furthermore, we identified Amer1 as binding partner of Amer3. Whereas Amer1 and Amer2 are linked to the plasma membrane by an N-terminal membrane localization domain, Amer3 lacks this domain. Amer3 localizes to the cytoplasm and nucleus of epithelial cells, and this is dependent on specific nuclear import and export sequences. Functionally, exogenous Amer3 enhances the expression of a β-catenin/T-cell factor-dependent reporter gene, and knockdown of endogenous Amer3 reduces Wnt target gene expression in colorectal cancer cells. Thus, Amer3 acts as an activator of Wnt signalling, in contrast to Amer1 and Amer2, which are inhibitors, suggesting a nonredundant role of Amer proteins in the regulation of this pathway. Our data, together with those of previous studies, provide a comprehensive picture of similarities and differences within the Amer protein family.

Cell–cell and cell–matrix interactions

In this year's annual ASCB Meeting Minisymposium on Cell–Cell and Cell–Matrix Interactions, new data were presented on several aspects of the biology of cell adhesions. The role of cell adhesions in cancer was the topic of three of the talks, two of which were focused on identifying how invasion is affected by cell–matrix and cell–cell adhesions. Yasmin Moshfegh from Louis Hodgson's lab (Albert Einstein College of Medicine) presented data showing a role for Rac1 in invadopodia formation, one of the initial steps in tumor cell invasion. The data showed that Rac1 suppresses invadopodia formation, and by using a fluorescence resonance energy transfer (FRET)-based Rac biosensor, the researchers showed that Rac1 activity is suppressed at sites of invadopodia formation. They further linked this to Rac1-mediated activation of PAK1, which in turn led to cortactin phosphorylation and invadopodia disassembly. Thomas Marshall (winner of the MBoC Paper of the Year Award) from Jody Rosenblatt's lab (Huntsman Cancer Institute) presented data on how APC (adenomatous polyposis coli) protein regulates the directionality of cell extrusion and how this could contribute to tumor cell metastasis in colon cancer, in which APC is frequently mutated. Cells with normal APC mainly extrude to the apical side of the tissue, whereas mutant APC reverses this directionality. The researchers also showed that the C-terminal domain of APC regulated this directionality. APC lacking this domain led to basal protrusion of the cancer cells, perhaps contributing to tumor cell invasion to basal layers. Taru Muranen from Joan Brugge's lab (Harvard Medical School) presented data on how extracellular matrix contact mediates drug resistance in tumor cells, wherein only matrix-attached cancer cells up-regulate a protective response (including anti-apoptotic Bcl-2 family proteins) in response to drug treatment, leading to cancer cell survival. In the other half of the Minisymposium, the speakers took a more mechanistic approach toward research on cell–matrix interactions. Viola Vogel (ETH Zurich) presented recent work on probes and new assays that demonstrate how cells exploit fibronectin fibrils as mechanochemical signal converters. She also presented proteomic data–derived evidence that extracellular phosphorylation and phosphorylated proteins are physiologically far more important than previously thought and are associated particularly with diseased tissues, such as those found in cancer. Michael Rubashkin from Valerie Weaver's lab (University of California–San Francisco) presented recent work on the development of a novel microscopy technique—scanning angle interference microscopy (SAIM)—that allows the study of focal adhesion structures in live cells at the nanoscale. SAIM was used to study how the relative position of vinculin changes in response to traction forces and how myosin contractility affects this localization. The researchers utilized vinculin activity mutants to study this and showed that active vinculin positions itself closer to the cell surface. This localization was also reflected in the structure of focal adhesions, suggesting that the relative position of vinculin corresponds to different adhesions. Caitlin Collins from Ellie Tzima's lab (University of North Carolina at Chapel Hill) reported on the role of the mechanosensory molecule PECAM-1 in endothelial mechanotransduction. The approach the researchers took was to use magnetic tweezers to apply tensional forces on PECAM-1 magnetic beads. On application of mechanical force on PECAM-1, they observed integrin and phosphatidylinositol 3-kinase pathway activation, both of which were required for adaptive stiffening of the endothelial cells. They further showed that this pathway led to global activation of RhoA and global formation of focal adhesions in endothelial cells. This pathway could have implications in vivo for atherosclerotic plaque formation, in which there is stiffening of the endothelial cells, and PECAM-1 could be employed to elicit some of the early events leading to plaque formation.

Crystal Structures of the Armadillo Repeat Domain of Adenomatous Polyposis Coli and Its Complex with the Tyrosine-Rich Domain of Sam68

Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm andSam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.

Messing up disorder: how do missense mutations in the tumor suppressor protein APC lead to cancer?

SummaryMutations in the adenomatous polyposis coli (APC) tumor suppressor gene strongly predispose to development of gastro-intestinal tumors. Central to the tumorigenic events in APC mutant cells is the uncontrolled stabilization and transcriptional activation of the protein β-catenin. Many questions remain as to how APC controls β-catenin degradation. Remarkably, the large C-terminal region of APC, which spans over 2000 amino acids and includes critical regions in downregulating β-catenin, is predicted to be natively unfolded. Here we discuss how this uncommonly large disordered region may help to coordinate the multiple cellular functions of APC. Recently, a significant number of germline and somatic missense mutations in the central region of APC were linked to tumorigenesis in the colon as well as extra-intestinal tissues. We classify and localize all currently known missense mutations in the APC structure. The molecular basis by which these mutations interfere with the function of APC remains unresolved. We propose several mechanisms by which cancer-related missense mutations in the large disordered domain of APC may interfere with tumor suppressor activity. Insight in the underlying molecular events will be invaluable in the development of novel strategies to counter dysregulated Wnt signaling by APC mutations in cancer.

Crystal structure of the armadillo repeat domain of adenomatous polyposis coli which reveals its inherent flexibility

The conserved armadillo repeat (ARM) domain of adenomatous polyposis coli (APC) protein plays an important role in the recognition of its binding partners. In this study, we report the crystal structure of APC-ARM (residues 407-775), which was determined to 2.9Å resolution. Our structure shows that the seven armadillo repeats of APC-ARM fold together into a compact domain, with Arm2 and Arm5 presenting some deviations from canonical armadillo repeats. There is a positively charged groove on the surface of APC-ARM, which might be the recognition site for APC-binding partners. Comparison of this structure with our previously reported structure of APC (407-751), together with normal mode analysis, reveals that the APC-ARM domain possesses a limited intrinsic flexibility. We propose that this intrinsic flexibility might be an inherent property of ARM domains in general.

Structural basis for the recognition of Asef by adenomatous polyposis coli

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers.

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal "basic" domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly-promoting factors with complementary activities.

-Catenin degradation mediated by the CID domain of APC provides a model for the selection of APC mutations in colorectal, desmoid and duodenal tumours

Biallelic mutation of the ADENOMATOUS POLYPOSIS COLI (APC) gene is a hallmark of sporadic colorectal cancer and colorectal, duodenal and desmoid tumours that develop in familial adenomatous polyposis (FAP) patients. The mutations affecting both APC alleles are interdependent, the position of the first APC mutation determining where the second hit will occur. This results in a complex pattern of mutation distribution in the APC sequence that translates into the stabilization of beta-catenin that in turn feeds the affected cells with a permanent mitogenic signal. We describe here a new APC domain, the beta-catenin inhibitory domain (CID) of APC located between the second and third 20 amino acid repeats and therefore present in many truncated APC products found in human tumours. In truncated APC, the CID is absolutely necessary to down-regulate the transcriptional activity and the level of beta-catenin, even when an axin/conductin binding site is present. The activity of the CID is dramatically reduced in several colon cancer cell lines and can be inhibited by shorter truncated APC lacking the CID. The CID is a direct target of the selective pressure acting on APC during tumourigenesis. It explains the interdependence of both APC mutations, not only in colorectal but also in duodenal and desmoid tumours.

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell–cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner — the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.

Biosensor-Based Micro-Affinity Purification for the Proteomic Analysis of Protein Complexes

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.