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.     

Axin-independent phosphorylation of APC controls beta-catenin signaling via cytoplasmic retention of beta-catenin

It has been shown that accumulation of free beta-catenin leads to mobility shift of adenomatous polyposis coli (APC) protein and that Axin facilitates this process. Here we show that the beta-catenin-mediated mobility shift of APC is due to phosphorylation of two domains of APC by casein kinase 1epsilon/glycogen synthase kinase 3beta and unknown kinase(s), respectively. Interestingly, our results suggest that this process does not require Axin. The phosphorylated APC showed higher affinity to beta-catenin in vivo, and fragments of APC containing the phosphorylated domains can inhibit beta-catenin/Tcf-mediated reporter activity regardless of their ability to reduce the level of beta-catenin. From our data we propose a new role of APC: accumulation of excessive cytoplasmic beta-catenin induces phosphorylation of APC and the phosphorylated APC retains beta-catenin in cytoplasm to prevent excessive beta-catenin signaling. The retained beta-catenin in cytoplasm by APC may be down-regulated by Axin 2, which is induced by beta-catenin/Tcf signaling.     

The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin

Adenomatous polyposis coli (APC) plays a critical role in the Wnt signaling pathway by tightly regulating beta-catenin turnover and localization. The central region of APC is responsible for APC-beta-catenin interactions through its seven 20 amino acid (20aa) repeats and three 15 amino acid (15aa) repeats. Using isothermal titration calorimetry, we have determined the binding affinities of beta-catenin with an APC 15aa repeat fragment and each of the seven 20aa repeats in both phosphorylated and unphosphorylated states. Despite sequence homology, different beta-catenin binding repeats of APC have dramatically different binding affinities with beta-catenin and thus may play different biological roles. The third 20aa repeat is by far the tightest binding site for beta-catenin among all the repeats. The fact that most APC mutations associated with colon cancers have lost the third 20aa repeat underlines the importance of APC-beta-catenin interaction in Wnt signaling and human diseases. For every 20aa repeat, phosphorylation dramatically increases its binding affinity for beta-catenin, suggesting phosphorylation has a critical regulatory role in APC function. In addition, our CD and NMR studies demonstrate that the central region of APC is unstructured in the absence of beta-catenin and Axin, and suggest that beta-catenin may interact with each of the APC 15aa and 20aa repeats independently.     

The ins and outs of APC and beta-catenin nuclear transport

Adenomatous polyposis coli (APC) and β-catenin, two key interacting proteins implicated in development and cancer, were recently found to traffic into and out of the nucleus in response to internal and external signals. The two proteins can enter and exit the nucleus independently, a discovery that has prompted debate about the previously proposed role of APC as a β-catenin chaperone. Here, we review the regulation of APC and β-catenin subcellular localization, in particular in cancer cells. We speculate that, in non-stimulated cells, APC actively exports β-catenin from the nucleus to the cytoplasm where its levels are regulated by degradation; and, conversely, that, in cancer cells or those stimulated by Wnt signaling, β-catenin degradation is inhibited and the accruing protein is capable of moving between the nucleus and cytoplasm independently of APC. Models that link APC and β-catenin transport to function are discussed.     

Down-regulation of beta-catenin by the colorectal tumor suppressor APC requires association with Axin and beta-catenin

The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. APC forms a complex with beta-catenin, Axin, and glycogen synthase kinase-3beta and induces the degradation of beta-catenin. In the present study, we examined whether APC association with Axin is required for degradation of beta-catenin. We found that a fragment of APC that induces beta-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Also, overexpression of an Axin fragment spanning the regulator of the G-protein signaling domain inhibited APC-mediated beta-catenin degradation. An APC fragment with mutated beta-catenin-binding sites but intact Axin-binding sites also failed to induce degradation of beta-catenin. These results suggest that APC requires interaction with Axin and beta-catenin to down-regulate beta-catenin.     

Cortical beta-catenin and APC regulate asymmetric nuclear beta-catenin localization during asymmetric cell division in C. elegans

In C. elegans, Wnt signaling regulates a number of asymmetric cell divisions. During telophase, WRM-1/beta-catenin localizes asymmetrically to the anterior cortex and the posterior daughter's nucleus. However, cortical WRM-1's functions are not known. Here, we use a membrane-targeted form of WRM-1 to show that cortical WRM-1 inhibits Wnt signaling and the nuclear localization of WRM-1. These functions are mediated by APR-1/APC, which regulates WRM-1 nuclear export. We also show that APR-1 as well as PRY-1/Axin and Dishevelled homologs localize asymmetrically to the cortex. Our results suggest a model in which cortical WRM-1 recruits APR-1 to the anterior cortex before and during division, and the cortical APR-1 stimulates WRM-1 export from the anterior nucleus at telophase. Because beta-catenin and APC are localized to the cortex in many cell types in different species, our results suggest that these cortical proteins may regulate asymmetric divisions or Wnt signaling in other organisms as well.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E- cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation

Beta-catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for beta-catenin degradation, and regulation of beta-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. Beta-catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb beta-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein beta-TrCP, a component of a ubiquitin ligase complex that mediates beta-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for beta-catenin phosphorylation by GSK3. Beta-catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in beta-catenin phosphorylation-degradation.     

Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.     

Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride–pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.