The Regulation Of Wnt Pathway β-catenin Phosphorylation And Nuclear/cytoplasmic Localization By Adenomatous Polyposis Coli Tumor Suppressor Gene APC

BackgroundWnt-β-catenin pathway is a major signaling route that controls embryonic patterning and tissue homeostasis. Its deregulation is involved in many cancers, which is in particular over-activated in virtually all colon cancer.The adenomatous polyposis coli (APC) tumor suppressor gene is a key negative regulator of the P-catenin signaling and its mutation could actually represent the initiating event for this type of cancer. Clinical studies have found that APC deletions or mutations, which could produce truncated APC protein and activate Wnt/β-catenin signaling pathway, occurred in70-80%of sporadic colorectal cancer. APC plays various roles in cell migration, chromosome separation and transcriptional regulation. On the one hand, APC involved in the P-catenin identification, phosphorylation and targeted degradation process; On the other hand, as a nuclear shuttle protein, APC might play a certain role in the nuclear translocation of β-catenin, thereby reducing the level of its nuclear and transcriptional activity.The pathway revolves around P-catenin and the regulation of β-catenin phosphorylation is the the central process, which is regulated by the destruction complex. However, the exact mechanism is still controversial. In one model, Axin and APC are thought to act as coordinate scaffolds that ensure the specificity of P-catenin phosphorylation and of its regulation by the Wnt pathway. Munemitsu et al. have shown that APC is also essential, since β-catenin accumulates when APC is mutated or depleted. In vitro experiments using pure recombinant proteins have indeed demonstrated that APC further increases the efficiency of the Axin-GSK3complex to phosphorylate β-catenin. Axin overexpression was found to rescue lower P-catenin signaling in APC-mutated cancer cells, indicating that APC may be dispensable when Axin levels are sufficiently high and Axin has been indeed considered to be the limiting factor in the pathway.The function of APC and the consequences of the mutations found in cancer cells remain unclear. So far, the regulation of P-catenin phosphorylation has only been studied in vitro, using purified proteins, or inferred from observation of steady state levels. It’s necessary to establish an assay to measure the endogenous kinase activity directly and clarify APC function on the regulation of β-catenin phosphorylation, which will be important to learn more about the mechanism of tumorgenesis.Based on the above questions, we compared the kinase activity of β-catenin in colon cancer SW480cells (which express a truncated APC at1338), SW480APC (cells rescued with full length APC, kindly gift from Antony Burgess). siRNA and Axin overexpression was also used for the investigation of APC role on β-catenin phosphorylation. Several β-catenin mutants [APC△15(R386A、APC△20(K345A, W383A)]was constructed and applied to analyze the specific domain. We used cell fraction for the further study on subcellular level. Besides, we also evaluate the role of APC in the regulation of p-catenin nuclear translocation in cells expressing physiological levels of wild type APC, truncated APC, or cells depleted of APC using the fluorescence recovery after photobleaching (FRAP) technology. Part1Regulation of wnt pathway P-catenin phosphorylatio by APC and tumor associated truncated APCObjective: To analyze the expression levels of Wnt pathway mainly related proteins and β-catenin phosphorylation activity in SW480cells and SW480APC cells, and further discuss the role of truncated APC and wild-type APC gene product in the regulation of β-catenin phosphorylation process.Methods:1. Western blot was used to detect the levels of APC, Axin, GSK3and CK1in SW480cells SW480APC total cell extracts.2. The kinase assay was performed to monitor the endogenous activity responsible for β-catenin phosphorylation in SW480cells and SW480APC total cell homogenates using recombinant β-catenin or mutant S45D p-catenin as substrate.3. APC siRNA interference was performed for both SW480and SW480APC cells, transient transfection of Axin was also used to analyze the phosphorylation activity of β-catenin.4. Using point-mutation technique, we constructed build APCA15(R386A), APCA20(K345A) and APCA20(W383A) three mutant p-catenin recombinant protein as a substrate for the kinase assay, and compared the phosphorylate activity of β-catenin with the wild-type β-catenin substrate.Results:1. Characterization of components of the Wnt pathway in SW480and SW480APC cells: SW480APC cells expressed relatively low levels of APC. The two cell lines expressed similar levels of Axin, GSK3and casein kinase1.2. We established a specific in vitro kinase assay to monitor the endogenous activity responsible for β-catenin phosphorylation. Using wild type β-catenin as substrate, the activity was significantly higher in SW480APC cells, both for Ser45and for Ser33/Ser37/Thr41(～five folds for Ser45,～two folds for Ser33/Ser37/Thr41). The kinase activity toward S45D β-catenin was also higher in SW480APC cell extracts.3. After the deletion of APC in SW480cells and SW480APC cells, both activities toward Ser45and Ser33/Ser37/Thr41were reduced in the two cell lines. The decrease was roughly proportional to the reduction in the levels of full length, respectively truncated APC. Mild Axin ectopic expression (2.3+/-0.4folds in parental SW480cells and2.5+/-0.9in SW480APC cell) failed to stimulate P-catenin phosphorylation, We also observed on the contrary a slight but reproducible decrease in Ser45phosphorylation in SW480cells.4. In all cases, the activity was lower than for wild type β-catenin. The difference was relatively mild for the mutant lacking binding to the15AA repeats, but quite strong for the two other mutants. Double345/383mutation led to a slight but not statistically significant decrease in the Ser33/Ser37/Thr41phosphorylation activity compared to the single mutants.Conclusions:1. APC could regulate the phosphorylation of β-catenin directly, and it is required for both phosphorylation steps.2. The APC mutation expressed in the parental SW480cells does not represent a complete loss-of-function and the resulting truncated APC has still a significant activity. Axin is not the limiting factor and cannot be substituted by increasing Axin levels.3. The15AA and20AA repeats, especially the only20AA repeat left in the truncated APC of SW480cells may play very important role in the P-catenin phosphorylation. Objective: To analyze the Wnt pathway main components and P-catenin phosphorylation activity in SW480cells and SW480APC cells and to clarify sublocalization of β-catenin phosphorylation.Methods:1. We used our newly established cell fractionation protocol to compare the distribution of the major components (APC, Axin, GSK3and CK1) of the pathway in SW480and SW480APC cells.2. We determined the subcellular distribution of β-catenin phosphorylation activity (pSer45and pSer33/Ser37/Thr41β-catenin) using the above-mentioned cell fractionation protocol.3. We performed rate zonal centrifugation on sucrose gradient to separate X fraction and detected the phosphorylation levels in different pools, the distribution of main components in Wnt pathway and also some subcellular markers like pericentrin, y-tubulin, Laminand LRP using Western Blot.4. We treated SW480and SW480APC cells with25mM LiCl for20min, and then shaked slowly with digitonin at4℃for10min before fixing. The co-localization among APC, CKla and pSer33/Ser37/Thr41β-catenin was observed by immunofluorescence compared the LiCl treated with cells and control cells.Results:1. We found that most components distributed in very similar patterns in parental and APC-rescued cells:APC and its truncated form were found in the cytosolic, nuclear insoluble and dense insoluble fractionsThe largest pool of Axin, casein kinase a and GSK3were found in the cytosol and fraction X. CKle were strongly enriched in fraction X.2. The most active pool was the dense insoluble fraction "X", which accounted for-60～70%of the total cell activity. Comparatively, the cytosol showed only a modest activity (10～20%). Nucleosol and membrane fractions displayed low to negligible activity. 3. The rate zonal centrifugation on Sucrose gradient resulted into13fractions(F1～F13), the most activity of β-catenin phosphorylation were found in the lower fractions enriched with the centrosomal markers pericentrin and y-tubulin.4. In samples from SW480APC extracts, Axin, CK1α and CK1ε showed prominent peaks in fractions8-10, thus co-sedimenting with both centrosomal markers and the kinase activity. The tightest correlation was found for Axin, CK1α and y-tubulin, all peaking in fraction9, where the kinase activity was maximal. GSK3mostly remained in the top of the gradient, but weak signal was found down to the dense fractions. In the case of paternal SW480cells, CKla also peaked in fraction9, but the other components were much more spread along the gradient.5. The confocal immunofluorescence results showed that both APC and CKla distributed in strikingly similar patterns, which were characterized by dense cytoplasmic accumulations, generally perinuclear. Among them, centrosomes, identified with γ-tubulin, were a prominent site of accumulation. We also detected very consistently accumulation of anti-pSer33/Ser37/Thr41β-catenin signal at centrosomes. The specificity of the signal for phosphorylated β-catenin was verified by comparing the signal in control and cells treated with the GSK3inhibitor LiCl.Conclusions:1. The main components involved in Wnt pathway focused on the Cs and X components, where consist with the main kinase activity of P-catenin phosphorylation.2. We improved the method to well separate centrosome and β-catenin phosphorylation activity co-sedimentation and partial co-localization with y-tubulin hints at a possible relationship with centrosomes. Part3Effects of APC and tumor associated truncated APC on the nuclear transport kinetics of β-catenin by FRAPObjective: To analyze the dynamic characteristics of β-catenin nuclear translocation, and to explore the impact of APC on the nuclear import and export of β-catenin.Methods:1. We did transit transfetion with YFP-β-catenin, GFP and Cherry-NLS plasmids and compare the nuclear/cytoplasm distribution in relative steady state after24-36h transfection. We performed FRAP experiments on SW480cells transfected with YFP-β-catenin and observe its kinetics. We analyzed all the data with two phase association fitting the curve.2. We analyzed the differences of β-catenin nuclear transport between SW480cell and SW480APC cell. APC siRNA was used to compare the dynamic changes between the APC depletion cells and the control cells.3. We treated SW480and SW480APC cells with the nuclear transport inhibitor LMB for4h or8h and analyze the nuclear translocation of β-catenin.Results:1. The nuclear signal was generally close to the cytoplasmic signal (median-1.2), although it varied from cell to cell. The ratio was largely similar for parental, APC-rescued and APC-depleted cells. We also verified that cell to cell variations were not related to levels of expression.2. The resulting recovery kinetics clearly fitted a two phase association model. The first phase of translocation was extremely rapidly in both direction (K～0.1/sec, half-life<10sec), the second an order of magnitude slower (K～0.01/sec, half-life>1min). The initial recovery phases of import and export was almost as fast as those measured for GFP, and the recovery approached a plateau around60-80%after5minutes. β-catenin appeared transported more efficiently than Cherry-NLS3. APC-rescued cells showed also differences: the fraction of the fast phase was halved compared to parental SW480cells, and the kinetics of the slow phase were decreased, with half live shifted from～1min to～5min, consistent with a more extensive retention by full length APC. As for export, expression of full length APC had no effect, but depletion again stimulated the process by increasing the fast moving fraction. The kinetics of the two phases were little affected by APC depletion, but the relative proportion of the phases was significantly changed, with an increased contribution of the fast phase and the final recovery set close to100%.4. The4-hours treatment had no detectable effect, neither on import nor on export. Longer treatments (8hrs) did show however an effect on import (but not export): Import was altogether increased, with the initial recovery phase becoming even faster than for APC-depleted cells, approaching the kinetics of free GFP.Conclusions:1. APC plays an important role in the nuclear transport of β-catenin, which could have an impact on β-catenin nuclear activities in normal and cancer cells.2. Wild type full length APC could induce β-catenin retention, which can slow down its nuclear import, while with no effect on the export. Truncated APC can accelerate the fast import phase, which is more obvious with APC depletion.