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.     

The miR-1-NOTCH3-Asef Pathway Is Important for Colorectal Tumor Cell Migration

The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC stimulates the activity of the Cdc42- and Rac1-specific guanine nucleotide exchange factor Asef and promotes the migration and invasion of colorectal tumor cells. Furthermore, Asef is overexpressed in colorectal tumors and is required for colorectal tumorigenesis. It is also known that NOTCH signaling plays critical roles in colorectal tumorigenesis and fate determination of intestinal progenitor cells. Here we show that NOTCH3 up-regulates Asef expression by activating the Asef promoter in colorectal tumor cells. Moreover, we demonstrate that microRNA-1 (miR-1) is down-regulated in colorectal tumors and that miR-1 has the potential to suppress NOTCH3 expression through direct binding to its 3’-UTR region. These results suggest that the miR-1-NOTCH3-Asef pathway is important for colorectal tumor cell migration and may be a promising molecular target for the treatment of colorectal tumors.     

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of β-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef^−/− mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.     

Adenomatous Polyposis Coli and Asef Function Downstream of Hepatocyte Growth Factor and Phosphatidylinositol 3-Kinase

Mutations of the tumor suppressor adenomatous polyposis coli (APC) are responsible for sporadic and familial colorectal tumors. APC negatively regulates Wnt signaling by inducing β-catenin degradation. It has also been shown that APC plays a role in the organization of cytoskeletal networks. APC interacts with Asef and Asef2, Rac1- and Cdc42-specific guanine nucleotide exchange factors (GEFs), and stimulates their GEF activity; thereby regulating cell morphology, adhesion, and migration. Truncated mutant APCs present in colorectal tumor cells activate Asef and Asef2 constitutively and contribute to their aberrant migratory properties. We show here that hepatocyte growth factor (HGF), as well as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), induce the accumulation and colocalization of APC and Asef in membrane ruffles and lamellipodia of epithelial cells. Both APC and Asef were found to be required for HGF-induced cell migration. Furthermore, we show that the effects of HGF, bFGF, and EGF on APC and Asef are mediated by the activation of phosphatidylinositol 3-kinase (PI3-kinase) and require the PH domain of Asef. These results suggest that Asef and APC function downstream of HGF and PI3-kinase, and play critical roles in growth factor-mediated regulation of cell morphology and migration.Mutations of the tumor suppressor gene adenomatous polyposis coli (APC)² are responsible for familial adenomatous polyposis (FAP), a dominantly inherited disease characterized by multiple adenomatous polyps in the colon (1, 2). The APC gene is also somatically mutated in the majority of sporadic colorectal tumors. The majority of the somatic mutations in APC is confined to its central region and result in the generation of truncated gene products. It is well known that APC induces degradation of β-catenin, a key Wnt signaling effector (3–6). Furthermore, it has recently been shown that APC also interacts with various other cellular proteins, including Asef, Asef2, IQGAP1, and kinesin-2, and regulates the organization of cytoskeletal networks, thereby controlling cell adhesion and motility (7–15).Asef is a guanine-nucleotide exchange factor (GEF) specific for Rac1 and Cdc42 (9–11, 15, 16). APC interacts via its armadillo repeat domain with an APC-binding region (ABR) in the NH₂ terminus of Asef. In addition to this ABR, Asef contains Dbl homology (DH), Pleckstrin homology (PH), and Src homology 3 (SH3) domains. The SH3 domain of Asef inhibits its own GEF activity by intramolecular binding to the DH domain (17, 18). The PH domain of Asef binds to phosphatidylinositol 3,4,5-trisphosphate (PIP3) and is required for its localization to the plasma membrane (19). APC enhances the GEF activity of Asef, presumably by relieving the intramolecular negative regulation and thereby regulates cell morphology, adhesion, and migration. A mutant form of Asef lacking the ABR shows strong GEF activity even in the absence of APC. Furthermore, truncated mutant APCs present in colorectal tumor cells activate Asef constitutively and cause increased aberrant migration. APC also activates Asef2, which has significant structural and functional similarities to Asef (11, 15). Thus, truncated mutant APCs, Asef and Asef2 may be important for adenoma formation as well as tumor progression to invasive malignancy.HGF is known to be important for embryonic development, wound healing, tissue regeneration, hematopoiesis, and tissue homeostasis (20, 21). The HGF receptor, which is encoded by the proto-oncogene c-met, is a tyrosine kinase, and its activation by HGF induces cell motility, invasion, and proliferation. Furthermore, HGF signaling is known to play a crucial role in tumor development and malignant progression, in particular by increasing tumor invasiveness and metastatic potential. Because the effects of APC-activated Asef on MDCK cells appear to be similar to those of HGF, we attempted to examine whether APC and Asef function downstream of HGF. In the present study, we show that APC and Asef indeed function downstream of HGF and that Asef is required for HGF-induced migration.     

PH domain-mediated membrane targeting of Asef

The APC-associated guanine nucleotide exchange factor (GEF) Asef regulates cell morphology and migration. Asef contains a pleckstrin homology (PH) domain in addition to Dbl homology (DH), APC-binding (ABR), and Src homology 3 (SH3) domains. Here we show that the PH domain of Asef binds to phosphatidylinositol 3,4,5-trisphophate [PtdIns(3,4,5)P3] and targets Asef to the cell-cell adhesion sites in MDCK II cells. Furthermore, we demonstrate that overexpression of Asef in MDCK II cells results in increases in the amounts of E-cadherin and the actin filaments at the sites of cell-cell contact. These results suggest that Asef is targeted via its PH domain to the cell-cell adhesion sites and is involved in the regulation of cell adhesion.     

The adenomatous polyposis coli‐associated exchange factors Asef and Asef2 are required for adenoma formation in ApcMin/+mice

Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)‐associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine‐nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in Apc^Min/+ mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC–Asef/Asef2 complex induces c‐Jun amino‐terminal kinase‐mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatement of colorectal tumours.     

Identification of a link between the tumour suppressor APC and the kinesin superfamily

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3 beta and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration.     

Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells.

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.     

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.     

Asef2 functions as a Cdc42 exchange factor and is stimulated by the release of an autoinhibitory module from a concealed C-terminal activation element

Asef (herein called Asef1) was identified as a Rac1-specific exchange factor stimulated by adenomatous polyposis coli (APC), contributing to colorectal cancer cell metastasis. We investigated Asef2, an Asef1 homologue having a similar N-terminal APC binding region (ABR) and Src-homology 3 (SH3) domain. Contrary to previous reports, we found that Asef1 and Asef2 exchange activity is Cdc42 specific. Moreover, the ABR of Asef2 did not function independently but acted in tandem with the SH3 domain to bind APC. The ABRSH3 also bound the C-terminal tail of Asef2, allowing it to function as an autoinhibitory module within the protein. Deletion of the C-terminal tail did not constitutively activate Asef2 as predicted; rather, a conserved C-terminal segment was required for augmented Cdc42 GDP/GTP exchange. Thus, Asef2 activation involves APC releasing the ABRSH3 from the C-terminal tail, resulting in Cdc42 exchange. These results highlight a novel exchange factor regulatory mechanism and establish Asef1 and Asef2 as Cdc42 exchange factors, providing a more appropriate context for understanding the contribution of APC in establishing cell polarity and migration.     

Coordinate recruitment of E-cadherin and ALCAM to cell-cell contacts by alpha-catenin

Here we report on the role of alpha-catenin in the cellular localization of activated leukocyte cell adhesion molecule, ALCAM, and cadherin-mediated cell adhesion in human prostate cancer cells. Cell lines that have a functional E-cadherin-mediated cell adhesion (DU-145 and LNCaP) show ALCAM staining at cell-cell contacts. In contrast, in cell lines that lack alpha-catenin expression (ALVA-31, PC-3, and PPC-1), E-cadherin-mediated adhesion is disturbed and ALCAM staining is cytoplasmic. A role of alpha-catenin in the recruitment of E-cadherin and ALCAM to cell-cell contacts was established by transfection of an alpha-N-catenin construct into cell lines ALVA-31 and PC-3. This resulted not only in the correct assembly of E-cadherin/alpha-catenin complexes at the cell membrane but also in localization of ALCAM to cell-cell contacts, indicating that indeed alpha-catenin affects ALCAM localization.     

Rho GTPase Cdc42 is a direct interacting partner of Adenomatous Polyposis Coli protein and can alter its cellular localization

Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC^1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC^1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins.     

Independent interactions of phosphorylated β-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions

Background

The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration.

Findings

In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions.

Conclusions

We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.     

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.     

A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin-mediated epithelial cell-cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell-cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors) did not inhibit cell-cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell-cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell-cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell-cell contacts and immediately initiate cell surface polarity.     

E-Cadherin Engagement Stimulates Tyrosine Phosphorylation

Cadherins are cell adhesion molecules concentrated at intercellular adherens junctions, where they form a multiprotein complex with cytoplasmic catenins. Although cell-cell interactions affect many aspects of cell behavior, little is known about signaling pathways triggered by cadherin engagement. We show here that E-cadherin-mediated cell-cell adhesion leads to a rapid increase in tyrosine phosphorylation at sites of cell-cell contact and that this stimulation of tyrosine phosphorylation can be mimicked by aggregation of E-cadherin with antibodies. The proteins that become phosphorylated are distinct from those previously shown to be tyrosine phosphorylated in response to integrin-mediated adhesion and include ras-GAP. We also find that E-cadherin-mediated tyrosine phosphorylation is not required for the assembly of adherens-type junctions.