结肠腺瘤样息肉基因APC对Wnt通路β-catenin磷酸化及其核浆转运的调节机制研究
|
摘要
研究背景
Wnt/β-catenin信号通路在调控胚胎发育和组织再生等过程中具有十分重要的作用,其异常激活与多种癌症,尤其是结直肠癌的发生密切相关。
结肠腺瘤样息肉(Adenomatous Polyposis Coli, APC)基因是Wnt/β-catenin信号通路的关键负向调节因子,其失活是结直肠癌发生的早期分子事件。临床研究发现,70-80%的散发性结直肠癌存在APC缺失或突变,产生截短型APC蛋白,激活Wnt/β-catenin信号通路,在细胞迁移、染色体分离和转录调节等方面具有多样性作用。一方面,APC参与β-catenin的识别、磷酸化和靶向降解过程；另一方面,APC可能作为核穿梭蛋白,在β-catenin的核转运过程中发挥某些特定作用,从而降低其核水平及转录活性。
β-catenin的磷酸化是Wnt通路的关键环节,降解复合体(destruction complex)是调节这一过程的重要因素,但其具体机制仍然存在着广泛的争议,经典模型认为,APC和Axin都作为支架蛋白,确保β-catenin磷酸化的特异性,并调控Wnt信号通路。Munemitsu等认为APC突变或缺失可导致β-catenin聚集,提出APC对β-catenin的磷酸化是必不可少的。此外,APC能够进一步增强Axin-GSK3复合体对磷酸化β-catenin的亲和力。但也有研究提出,当细胞中Axin水平足够高时,APC的作用价值不大,Axin是Wnt通路的限制因素。尽管学者们对APC在Wnt通路中的作用等方面进行了大量研究,但APC的生物学功能至今仍然存在着很多未知的方面,且目前研究大多是在β-catenin稳定表达状态下进行的,有必要建立激酶实验方法直接检测内源性β-catenin的磷酸化活性,进一步明确APC或截短型APC在Wnt通路中对β-catenin磷酸化的调节作用,其对了解结肠癌等肿瘤的发病机制具有重要意义。
基于上述问题,本课题通过激酶检测实验比较SW480(APC第1338个密码子发生突变)和SW480APC(稳定转染全长APC的SW480APC细胞株,由Antony Burgess馈赠)两种细胞株中β-catenin的激酶活性,应用siRNA干扰和Axin过表达等方法初步探讨了APC在β-catenin磷酸化过程中的作用,利用点突变技术构建APC△15(R386A)、APC△20(K345A, W383A)突变型β-catenin重组蛋白,分析APC发挥作用的具体结构区域。并采用细胞分级等方法从亚细胞水平阐述APC对Wnt通路β-catenin磷酸化调节的作用,明确其发生部位。此外,本研究还应用荧光漂白后恢复(FRAP)技术研究β-catenin核转运的动力学特征以及APC在其中可能发挥的作用,为进一步研究Wnt通路提供一定的依据。
第一部分APC和肿瘤相关截短型APC对Wnt通路β-catenin磷酸化的调节作用
目的：分析SW480细胞和SW480APC细胞中Wnt通路主要相关蛋白的表达情况及β-catenin磷酸化活性,探讨截短型APC和野生型APC基因产物在β-catenin磷酸化过程中的调节作用。
方法：
1.Western blot方法检测SW480细胞和SW480APC细胞总提取物中APC、Axin、GSK3和CK1的表达情况。
2.分别以野生型β-catenin和突变型S45D β-catenin为底物进行激酶实验,检测磷酸化β-catenin蛋白(pSer45和pSer33/Ser37/Thr41)在SW480细胞和SW480APC细胞中的激酶活性水平。
3.应用WC siRNA干扰和瞬时转染Axin技术,激酶实验检测β-catenin在APC缺失或Axin过表达情况下的磷酸化活性。
4.利用点突变技术,构建APC△15(R386A)、APC△20(K345A)和APC△20(W383A)三种突变型β-catenin重组蛋白作为底物进行激酶实验,并与野生型β-catenin底物比较,检测磷酸化β-catenin的变化情况。
结果：
1.SW480和SW480APC细胞中Wnt通路组成特性：除SW480APC细胞中可见明显的低表达量的全长APC外,其它蛋白如Axin、GSK3和CK1等的表达量基本相同。
2.本研究建立了直接检测结肠癌细胞株SW480细胞中内源性P-catenin磷酸化活性的激酶实验方法。发现以野生型P-catenin为底物时,SW480APC细胞中两种磷酸化P-catenin (pSer45和pSer33/Ser37/Thr41)的水平显著高于SW480细胞,其中pSer45β-catenin水平升高5倍,pSer33/Ser37/Thr41β-catenin水平升高2倍；以S45D β-catenin为底物进行激酶实验的结果表明,SW480APC细胞中pSer33/Ser37/Thr41β-catenin活性也明显高于SW480细胞的磷酸化活性。
3.利用siRNA敲除APC后,两种细胞株中p-catenin的激酶活性均显著降低,其降低程度与全长APC缺失程度,特别是截短APC缺失程度相关。中度Axin异位表达(SW480细胞中2.3倍,SW480APC细胞中2.5倍)未能显著刺激β-catenin磷酸化水平增高,且SW480细胞中可见Ser45β-catenin活性轻度降低。
4.与野生型β-catenin底物相比,以三种突变型β-catenin重组蛋白为底物进行激酶实验,其β-catenin磷酸化活性均降低,并显著见于APC△20(K345A)和APC△20(W383A) β-catenin突变体,在APC△15(R386A) β-catenin突变体中的磷酸化活性减弱程度相对较轻。
结论：
1.本研究建立了直接检测结肠癌细胞株SW480细胞中内源性β-catenin磷酸化活性的激酶实验方法。发现APC可直接调节Wnt通路β-catenin的磷酸化过程,并且参与CK1及GSK3介导的两个磷酸化阶段。
2.SW480细胞中截短APC片段仍能在β-catenin磷酸化过程中发挥较大的作用。Axin并不是β-catenin磷酸化的限制因素,过量表达Axin并不能补偿APC的调节作用。
3.证明了APC结构中的15和20氨基酸重复序列,特别是第一个20氨基酸重复序列结构在β-catenin磷酸化过程中发挥重要的作用。
目的：分析SW480细胞和SW480APC细胞中Wnt通路降解复合体主要组成蛋白及β-catenin磷酸化活性的亚细胞定位,进一步探讨β-catenin磷酸化的发生部位。
方法：
1.利用细胞分级方法,检测SW480和SW480APC细胞中Wnt通路降解复合体主要组成蛋白APC、Axin、GSK3和CKl等在细胞浆(Cs)、细胞核可溶成分(Ns)和细胞核不可溶成分(Ni)、膜类成分(M)及由细胞核和细胞骨架等成分组成的致密不可溶性混合物成分(X)中的表达情况。
2.分别以野生型和S45D突变型β-catenin为底物,对各亚细胞组分进行激酶实验,Western Blot检测pS45和pS33/S37/T41β-catenin水平。
3.应用蔗糖密度区带离心方法进一步分离X组分,激酶实验检测不同密度区带离心后各组分中β-catenin的磷酸化水平。Western Blot检测各组分中APC、Axin、 GSK3、CK1α、CK1ε及相关亚细胞结构标志物pericentrin、γ-tubulin、Lamin、LRP等的分布情况。
4.SW480和SW480APC细胞经25mM LiCl培养20min后,加入洋地黄皂苷4℃C缓慢摇10min后固定,免疫荧光分别观察LiCl处理组细胞和对照组细胞内APC、CK1αt和pS33/S37/T41β-catenin的共定位情况。
结果：
1.两种细胞株亚细胞结构中的分布模式基本一致。全长APC及其截短片段可见于Cs、Ni和X组分中,但主要仍集中于Cs中。Axin、CK1α和GSK3主要分布于Cs和X中,而CKlε主要分布于X组分中。
2. β-catenin磷酸化活性主要发生在X组分中(约占总活性的60-70%),Cs中的活性相对较弱(仅为10～20%),Ni中磷酸化β-catenin程度接近Cs水平；Ns或M组分中活性很低或未见β-catenin的磷酸化。
3.X组分经蔗糖密度区带离心后,在沉降池内从液面到底部可分为13个不同的密度区带(F1-F13)。激酶活性主要见于下层高密度组分中(F8-F10),Western Blot证实其与中心体富集区域相关。
4. SW480APC细胞中,APC、Axin、CK1α和CK1ε主要分布于第F8-F10组分中,这与中心体定位和激酶活性发生部位一致。GSK3主要集中于低密度组分中,少量可见于底部高密度区域。而在SW480细胞中,可见CKla高表达于F9组分,其他各蛋白则呈弥散分布。
5.共聚焦显微镜免疫荧光结果显示：APC与CKla共定位于细胞核周边的胞浆中,γ-tubuli染色结果表明该区域与中心体定位一致,并发现pS33/S37/T41β-catenin也在此处表达。应用GSK3抑制剂LiCl处理细胞后免疫荧光结果进一步确认了pS33/S37/T41β-catenin的中心体定位。
结论：
1.Wnt通路降解复合体主要组成蛋白(APC、Axin、GSK和CK)主要集中于Cs和X组分中,其分布与β-catenin磷酸化活性发生部位一致。
2.完善建立了可用于分离SW480细胞中心体的方法,证实了中心体是β-catenin磷酸化活性发生的关键部位,β-catenin的磷酸化与以中心体相关的共沉淀复合物形成相关。
目的：研究β-catenin核转运的动力学特征,探讨APC对β-catenin入核和出核运动的影响。方法：
1.瞬时转染YFP-β-catenin、GFP、Cherry-NLS质粒24-36h后,比较β-catenin在核内和胞浆内的相对稳态分布情况。应用FRAP技术观察并检测这些荧光蛋白在SW480和SW480APC细胞内的分布和核内运动情况。应用二元扩散经验方程对所得数据进行拟合曲线分析。
2.比较SW480细胞和SW480APC细胞中β-catenin的核转运过程。应用APCsiRNA转染SW480细胞24-36h后,比较APC敲除组细胞与对照组细胞内YFP-β-catenin核转运的动力学变化。
3.SW480和SW480APC细胞转染YFP-β-catenin24h后,应用出核转运抑制剂LMB分别预处理细胞4h和8h, FRAP观察β-catenin的核转运变化情况。
结果：
1. GFP、Cherry-NLS及不同条件下的YFP-β-catenin的核/浆比例相近,(中位数～1.2)。含不同β-catenin表达水平的各个细胞间差异不影响其荧光恢复的动力学特征。
2.SW480细胞的动力学特征符合二元扩散经验方程,即早期快速运动时相(K～0.1/sec,tl/2<10s)和后期相对缓慢的恢复时相(迁移速率K～0.01/sec,t1a>lmin)。其中,早期快速时相的扩散运动与野生型全长APC或截短APC无显著相关性。SW480细胞中β-catenin入核和出核的早期运动时相速度几乎与GFP表现一致,但5min后达到平台期时仅能恢复60-80%。β-catenin的核转运速度快于Cherry-NLS的表现。
3.与SW480细胞相比,β-catenin在SW480APC细胞中的慢性恢复时相期的动力学降低,其半衰期时间从1min到5min不等,这与全长APC导致β-catenin广泛停留相关。对于β-catenin的核输出,全长APC的表达对其并无任何影响,但截短APC能够加速早期快速移动时相过程。比较SW480和APC敲除后SW480细胞的核转运功能,发现APC敲除后,各阶段的相对比例显著改变,表现为快速时相期的增快和后期荧光恢复程度增强。
4.LMB作用4h后,β-catenin的入核与出核运动未见明显改变。LMB处理8h后,可见β-catenin入核运动显著加快,早期移动时相高于APC敲除细胞,几乎与GFP的动力学变化一致；LMB对β-catenin的出核过程无影响。
结论：
1.本研究证实了APC作为核穿梭蛋白,在β-catenin的核转运过程中具有重要作用,可能影响肿瘤细胞中β-catenin的核内功能,这对进一步研究Wnt/β-catenin通路提供一定的依据。
2.野生型全长APC可引起β-catenin滞留,导致β-catenin入核运动减慢,但对其出核运动无明显改变。截短APC能够加速β-catenin早期快速入核过程,这一变化在APC缺失时更为明显,表明截短APC对β-catenin入核和出核运动的重要影响。
Background
Wnt-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 APC
Objective: 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 FRAP
Objective: 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-NLS
3. 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.
Wnt/β-catenin信号通路在调控胚胎发育和组织再生等过程中具有十分重要的作用,其异常激活与多种癌症,尤其是结直肠癌的发生密切相关。
结肠腺瘤样息肉(Adenomatous Polyposis Coli, APC)基因是Wnt/β-catenin信号通路的关键负向调节因子,其失活是结直肠癌发生的早期分子事件。临床研究发现,70-80%的散发性结直肠癌存在APC缺失或突变,产生截短型APC蛋白,激活Wnt/β-catenin信号通路,在细胞迁移、染色体分离和转录调节等方面具有多样性作用。一方面,APC参与β-catenin的识别、磷酸化和靶向降解过程；另一方面,APC可能作为核穿梭蛋白,在β-catenin的核转运过程中发挥某些特定作用,从而降低其核水平及转录活性。
β-catenin的磷酸化是Wnt通路的关键环节,降解复合体(destruction complex)是调节这一过程的重要因素,但其具体机制仍然存在着广泛的争议,经典模型认为,APC和Axin都作为支架蛋白,确保β-catenin磷酸化的特异性,并调控Wnt信号通路。Munemitsu等认为APC突变或缺失可导致β-catenin聚集,提出APC对β-catenin的磷酸化是必不可少的。此外,APC能够进一步增强Axin-GSK3复合体对磷酸化β-catenin的亲和力。但也有研究提出,当细胞中Axin水平足够高时,APC的作用价值不大,Axin是Wnt通路的限制因素。尽管学者们对APC在Wnt通路中的作用等方面进行了大量研究,但APC的生物学功能至今仍然存在着很多未知的方面,且目前研究大多是在β-catenin稳定表达状态下进行的,有必要建立激酶实验方法直接检测内源性β-catenin的磷酸化活性,进一步明确APC或截短型APC在Wnt通路中对β-catenin磷酸化的调节作用,其对了解结肠癌等肿瘤的发病机制具有重要意义。
基于上述问题,本课题通过激酶检测实验比较SW480(APC第1338个密码子发生突变)和SW480APC(稳定转染全长APC的SW480APC细胞株,由Antony Burgess馈赠)两种细胞株中β-catenin的激酶活性,应用siRNA干扰和Axin过表达等方法初步探讨了APC在β-catenin磷酸化过程中的作用,利用点突变技术构建APC△15(R386A)、APC△20(K345A, W383A)突变型β-catenin重组蛋白,分析APC发挥作用的具体结构区域。并采用细胞分级等方法从亚细胞水平阐述APC对Wnt通路β-catenin磷酸化调节的作用,明确其发生部位。此外,本研究还应用荧光漂白后恢复(FRAP)技术研究β-catenin核转运的动力学特征以及APC在其中可能发挥的作用,为进一步研究Wnt通路提供一定的依据。
第一部分APC和肿瘤相关截短型APC对Wnt通路β-catenin磷酸化的调节作用
目的：分析SW480细胞和SW480APC细胞中Wnt通路主要相关蛋白的表达情况及β-catenin磷酸化活性,探讨截短型APC和野生型APC基因产物在β-catenin磷酸化过程中的调节作用。
方法：
1.Western blot方法检测SW480细胞和SW480APC细胞总提取物中APC、Axin、GSK3和CK1的表达情况。
2.分别以野生型β-catenin和突变型S45D β-catenin为底物进行激酶实验,检测磷酸化β-catenin蛋白(pSer45和pSer33/Ser37/Thr41)在SW480细胞和SW480APC细胞中的激酶活性水平。
3.应用WC siRNA干扰和瞬时转染Axin技术,激酶实验检测β-catenin在APC缺失或Axin过表达情况下的磷酸化活性。
4.利用点突变技术,构建APC△15(R386A)、APC△20(K345A)和APC△20(W383A)三种突变型β-catenin重组蛋白作为底物进行激酶实验,并与野生型β-catenin底物比较,检测磷酸化β-catenin的变化情况。
结果：
1.SW480和SW480APC细胞中Wnt通路组成特性：除SW480APC细胞中可见明显的低表达量的全长APC外,其它蛋白如Axin、GSK3和CK1等的表达量基本相同。
2.本研究建立了直接检测结肠癌细胞株SW480细胞中内源性P-catenin磷酸化活性的激酶实验方法。发现以野生型P-catenin为底物时,SW480APC细胞中两种磷酸化P-catenin (pSer45和pSer33/Ser37/Thr41)的水平显著高于SW480细胞,其中pSer45β-catenin水平升高5倍,pSer33/Ser37/Thr41β-catenin水平升高2倍；以S45D β-catenin为底物进行激酶实验的结果表明,SW480APC细胞中pSer33/Ser37/Thr41β-catenin活性也明显高于SW480细胞的磷酸化活性。
3.利用siRNA敲除APC后,两种细胞株中p-catenin的激酶活性均显著降低,其降低程度与全长APC缺失程度,特别是截短APC缺失程度相关。中度Axin异位表达(SW480细胞中2.3倍,SW480APC细胞中2.5倍)未能显著刺激β-catenin磷酸化水平增高,且SW480细胞中可见Ser45β-catenin活性轻度降低。
4.与野生型β-catenin底物相比,以三种突变型β-catenin重组蛋白为底物进行激酶实验,其β-catenin磷酸化活性均降低,并显著见于APC△20(K345A)和APC△20(W383A) β-catenin突变体,在APC△15(R386A) β-catenin突变体中的磷酸化活性减弱程度相对较轻。
结论：
1.本研究建立了直接检测结肠癌细胞株SW480细胞中内源性β-catenin磷酸化活性的激酶实验方法。发现APC可直接调节Wnt通路β-catenin的磷酸化过程,并且参与CK1及GSK3介导的两个磷酸化阶段。
2.SW480细胞中截短APC片段仍能在β-catenin磷酸化过程中发挥较大的作用。Axin并不是β-catenin磷酸化的限制因素,过量表达Axin并不能补偿APC的调节作用。
3.证明了APC结构中的15和20氨基酸重复序列,特别是第一个20氨基酸重复序列结构在β-catenin磷酸化过程中发挥重要的作用。
目的：分析SW480细胞和SW480APC细胞中Wnt通路降解复合体主要组成蛋白及β-catenin磷酸化活性的亚细胞定位,进一步探讨β-catenin磷酸化的发生部位。
方法：
1.利用细胞分级方法,检测SW480和SW480APC细胞中Wnt通路降解复合体主要组成蛋白APC、Axin、GSK3和CKl等在细胞浆(Cs)、细胞核可溶成分(Ns)和细胞核不可溶成分(Ni)、膜类成分(M)及由细胞核和细胞骨架等成分组成的致密不可溶性混合物成分(X)中的表达情况。
2.分别以野生型和S45D突变型β-catenin为底物,对各亚细胞组分进行激酶实验,Western Blot检测pS45和pS33/S37/T41β-catenin水平。
3.应用蔗糖密度区带离心方法进一步分离X组分,激酶实验检测不同密度区带离心后各组分中β-catenin的磷酸化水平。Western Blot检测各组分中APC、Axin、 GSK3、CK1α、CK1ε及相关亚细胞结构标志物pericentrin、γ-tubulin、Lamin、LRP等的分布情况。
4.SW480和SW480APC细胞经25mM LiCl培养20min后,加入洋地黄皂苷4℃C缓慢摇10min后固定,免疫荧光分别观察LiCl处理组细胞和对照组细胞内APC、CK1αt和pS33/S37/T41β-catenin的共定位情况。
结果：
1.两种细胞株亚细胞结构中的分布模式基本一致。全长APC及其截短片段可见于Cs、Ni和X组分中,但主要仍集中于Cs中。Axin、CK1α和GSK3主要分布于Cs和X中,而CKlε主要分布于X组分中。
2. β-catenin磷酸化活性主要发生在X组分中(约占总活性的60-70%),Cs中的活性相对较弱(仅为10～20%),Ni中磷酸化β-catenin程度接近Cs水平；Ns或M组分中活性很低或未见β-catenin的磷酸化。
3.X组分经蔗糖密度区带离心后,在沉降池内从液面到底部可分为13个不同的密度区带(F1-F13)。激酶活性主要见于下层高密度组分中(F8-F10),Western Blot证实其与中心体富集区域相关。
4. SW480APC细胞中,APC、Axin、CK1α和CK1ε主要分布于第F8-F10组分中,这与中心体定位和激酶活性发生部位一致。GSK3主要集中于低密度组分中,少量可见于底部高密度区域。而在SW480细胞中,可见CKla高表达于F9组分,其他各蛋白则呈弥散分布。
5.共聚焦显微镜免疫荧光结果显示：APC与CKla共定位于细胞核周边的胞浆中,γ-tubuli染色结果表明该区域与中心体定位一致,并发现pS33/S37/T41β-catenin也在此处表达。应用GSK3抑制剂LiCl处理细胞后免疫荧光结果进一步确认了pS33/S37/T41β-catenin的中心体定位。
结论：
1.Wnt通路降解复合体主要组成蛋白(APC、Axin、GSK和CK)主要集中于Cs和X组分中,其分布与β-catenin磷酸化活性发生部位一致。
2.完善建立了可用于分离SW480细胞中心体的方法,证实了中心体是β-catenin磷酸化活性发生的关键部位,β-catenin的磷酸化与以中心体相关的共沉淀复合物形成相关。
目的：研究β-catenin核转运的动力学特征,探讨APC对β-catenin入核和出核运动的影响。方法：
1.瞬时转染YFP-β-catenin、GFP、Cherry-NLS质粒24-36h后,比较β-catenin在核内和胞浆内的相对稳态分布情况。应用FRAP技术观察并检测这些荧光蛋白在SW480和SW480APC细胞内的分布和核内运动情况。应用二元扩散经验方程对所得数据进行拟合曲线分析。
2.比较SW480细胞和SW480APC细胞中β-catenin的核转运过程。应用APCsiRNA转染SW480细胞24-36h后,比较APC敲除组细胞与对照组细胞内YFP-β-catenin核转运的动力学变化。
3.SW480和SW480APC细胞转染YFP-β-catenin24h后,应用出核转运抑制剂LMB分别预处理细胞4h和8h, FRAP观察β-catenin的核转运变化情况。
结果：
1. GFP、Cherry-NLS及不同条件下的YFP-β-catenin的核/浆比例相近,(中位数～1.2)。含不同β-catenin表达水平的各个细胞间差异不影响其荧光恢复的动力学特征。
2.SW480细胞的动力学特征符合二元扩散经验方程,即早期快速运动时相(K～0.1/sec,tl/2<10s)和后期相对缓慢的恢复时相(迁移速率K～0.01/sec,t1a>lmin)。其中,早期快速时相的扩散运动与野生型全长APC或截短APC无显著相关性。SW480细胞中β-catenin入核和出核的早期运动时相速度几乎与GFP表现一致,但5min后达到平台期时仅能恢复60-80%。β-catenin的核转运速度快于Cherry-NLS的表现。
3.与SW480细胞相比,β-catenin在SW480APC细胞中的慢性恢复时相期的动力学降低,其半衰期时间从1min到5min不等,这与全长APC导致β-catenin广泛停留相关。对于β-catenin的核输出,全长APC的表达对其并无任何影响,但截短APC能够加速早期快速移动时相过程。比较SW480和APC敲除后SW480细胞的核转运功能,发现APC敲除后,各阶段的相对比例显著改变,表现为快速时相期的增快和后期荧光恢复程度增强。
4.LMB作用4h后,β-catenin的入核与出核运动未见明显改变。LMB处理8h后,可见β-catenin入核运动显著加快,早期移动时相高于APC敲除细胞,几乎与GFP的动力学变化一致；LMB对β-catenin的出核过程无影响。
结论：
1.本研究证实了APC作为核穿梭蛋白,在β-catenin的核转运过程中具有重要作用,可能影响肿瘤细胞中β-catenin的核内功能,这对进一步研究Wnt/β-catenin通路提供一定的依据。
2.野生型全长APC可引起β-catenin滞留,导致β-catenin入核运动减慢,但对其出核运动无明显改变。截短APC能够加速β-catenin早期快速入核过程,这一变化在APC缺失时更为明显,表明截短APC对β-catenin入核和出核运动的重要影响。
Background
Wnt-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 APC
Objective: 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 FRAP
Objective: 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-NLS
3. 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.
引文
1. Logan, C.Y. and R. Nusse. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol.2004;20:781-810.
2. Moon RT, Kohn AD, De Ferrari GV, and Kaykas A. WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet.2004;5(9):691-701.
3. Reya, T. and H. Clevers, Wnt signalling in stem cells and cancer. Nature.2005;434(7035):843-50.
4. Giles, R.H., J.H. van Es, and H. Clevers. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta.2003;653(1):1-24.
5. Polakis, P. Wnt signaling and cancer. Genes Dev.2000;14(15):1837-51.
6. Moon, R.T., J.D. Brown, and M. Torres. WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 1997; 13(4):157-62.
7. Wodarz, A. and R. Nusse. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol.1998;14:59-88.
8. Galea, M.A., A. Eleftheriou, and B.R. Henderson. ARM domain-dependent nuclear import of adenomatous polyposis coli protein is stimulated by the B56 alpha subunit of protein phosphatase 2A. J Biol Chem.2001;276(49):45833-9.
9. Neufeld KL, Nix DA, Bogerd H, Kang Y, Beckerle MC, Cullen BR, and White RL. Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. Proc Natl Acad Sci U S A.2000;97(22):12085-90.
10. Zeng L, Fagotto F, Zhang T, Hsu W, Vasicek TJ, Lee JJ, Tilghman SM, Gumbiner BM, and Costantini F. The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell.1997;90(1):181-92.
11. Kikuchi, A. Roles of Axin in the Wnt signalling pathway. Cell Signal.1999; 11(11):777-88.
12. Fiedler M, Mendoza-Topaz C, Rutherford TJ, Mieszczanek J, and Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating beta-catenin. Proc Natl Acad Sci U S A. 2011;108(5):1937-42.
13. Sakanaka, C. and L.T. Williams. Functional domains of axin. Importance of the C terminus as an oligomerization domain. J Biol Chem.1999;274(20):14090-3.
14. Huber, A.H., W.J. Nelson, and W.I. Weis, Three-dimensional structure of the armadillo repeat region of beta-catenin. Cell. 1997;90(5):871-82.
15. Huber, A.H. and W.I. Weis, The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin. Cell.2001;105(3):391-402.
16. Graham TA, Weaver C, Mao F, Kimelman D, and Xu W. Crystal structure of a beta-catenin/Tcf complex. Cell.2000; 103(6):885-96.
17. Ha NC, Tonozuka T, Stamos JL, Choi HJ, and Weis WI. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell.2004;15(4):511-21.
18. Pokutta, S. and W.I. Weis, The cytoplasmic face of cell contact sites. Curr Opin Struct Biol.2002;12(2):255-62.
19. von Kries JP, Winbeck G, Asbrand C, Schwarz-Romond T, Sochnikova N, Dell'Oro A, Behrens J, and Birchmeier W. Hot spots in beta-catenin for interactions with LEF-1, conductin and APC. Nat Struct Biol.2000;7(9):800-7.
20. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, and He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell.2002;108(6):837-47.
21. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, and Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45:a molecular switch for the Wnt pathway. Genes Dev.2002; 16(9):1066-76.
22. Yanagawa S, Matsuda Y, Lee JS, Matsubayashi H, Sese S, Kadowaki T, and Ishimoto A. Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila. Embo J.2002;21(7):1733-42.
23. Peters JM, McKay RM, McKay JP, Graff JM. Peters JM, McKay RM, McKay JP, and Graff JM. Casein kinase I transduces Wnt signals. Nature.1999;401(6751):345-50.
24. Tickenbrock L, Kossmeier K, Rehmann H, Herrmann C, and Miiller O. Differences between the interaction of beta-catenin with non-phosphorylated and single-mimicked phosphorylated 20-amino acid residue repeats of the APC protein. J Mol Biol.2003;327(2):359-67.
25. Rubinfeld, B., D.A. Tice, and P. Polakis, Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase lepsilon. J Biol Chem.2001;276(42):39037-45.
26. Lee, E., A. Salic, and M.W. Kirschner, Physiological regulation of [beta]-catenin stability by Tcf3 and CKlepsilon. J Cell Biol.2001;154(5):983-93.
27. Gao ZH, Seeling JM, Hill V, Yochum A, and Virshup DM. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci U S A.2002;99(3):1182-7.
28. Yost C, Torres M, Miller JR, Huang E, Kimelman D, and Moon RT. The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev.1996;10(12):1443-54.
29. Dajani R, Fraser E, Roe SM, Yeo M, Good VM, Thompson V, Dale TC, Pearl LH. Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. Embo J.2003;22(3):494-501.
30. Bhanot P, Fish M, Jemison JA, Nusse R, Nathans J, and Cadigan KM. Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. Development. 1999;126(18):4175-86..
31. Willert K, Logan CY, Arora A, Fish M, and Nusse R. A Drosophila Axin homolog, Daxin, inhibits Wnt signaling. Development. 1999;126(18):4165-73.
32. Willert, K., S. Shibamoto, and R. Nusse, Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex. Genes Dev.1999; 13(14):1768-73.
33. Kobayashi M, Honma T, Matsuda Y, Suzuki Y, Narisawa R, Ajioka Y, and Asakura H. Nuclear translocation of beta-catenin in colorectal cancer. Br J Cancer.2000;82(10):1689-93.
34. Maruyama K, Ochiai A, Akimoto S, Nakamura S, Baba S, Moriya Y, and Hirohashi S. Cytoplasmic beta-catenin accumulation as a predictor of hematogenous metastasis in human colorectal cancer. Oncology.2000;59(4):302-9.
35. Munemitsu S, Albert I, Souza B, Rubinfeld B, and Polakis P. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci U S A, 1995.92(7):3046-50.
36. Fagotto F, Jho Eh, Zeng L, Kurth T, Joos T, Kaufmann C, and Costantini F. Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization. J Cell Biol.1999; 145(4):741-56.
37. Hart MJ, de los Santos R, Albert IN, Rubinfeld B, and Polakis P. Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. Curr Biol.1998;8(10):573-81.
38. Hinoi T, Yamamoto H, Kishida M, Takada S, Kishida S, Kikuchi A. Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin. J Biol Chem.2000;275(44):34399-406.
39. Kishida S, Yamamoto H, Ikeda S, Kishida M, Sakamoto I, Koyama S, and Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem.1998;273(18):10823-6.
40. Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, Masiarz FR, Munemitsu S, Polakis P. Association of the APC gene product with beta-catenin. Science. 1993;262(5140):1731-4.
41. Su, L.K., B. Vogelstein, and K.W. Kinzler, Association of the APC tumor suppressor protein with catenins. Science. 1993;262(5140):1734-7.
42. Kimelman, D. and W. Xu, beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene.2006;25(57):7482-91.
43. Bienz, M., The subcellular destinations of APC proteins. Nat Rev Mol Cell Biol.2002;3(5):328-38.
44. Su Y, Fu C, Ishikawa S, Stella A, Kojima M, Shitoh K, Schreiber EM, Day BW, and Liu B. APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase. Mol Cell.2008;32(5):652-61.
45. Li VS, Ng SS, Boersema PJ, Low TY, Karthaus WR, Gerlach JP, Mohammed S, Heck AJ, Maurice MM, Mahmoudi T, and Clevers H. Wnt signaling through inhibition of beta-catenin degradation in an intact Axinl complex. Cell.2012;149(6):1245-56.
46. Roberts DM, Pronobis MI, Poulton JS, Waldmann JD, Stephenson EM, Hanna S, and Peifer M. Deconstructing the sscatenin destruction complex:mechanistic roles for the tumor suppressor APC in regulating Wnt signaling. Mol Biol Cell.2011;22(11):845-63.
47. Faux MC, Coates JL, Kershaw NJ, Layton MJ, and Burgess AW. Independent interactions of phosphorylated beta-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions. PLoS One. 5(11):e14127.
48. Stambolic, V., L. Ruel, and J.R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol.1996;6(12):1664-8.
49. Taelman VF, Dobrowolski R, Plouhinec JL, Fuentealba LC, Vorwald PP, Gumper I, Sabatini DD, and De Robertis EM. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell.2010;143(7):1136-48.
50. Lee E, Salic A, Kruger R, Heinrich R, and Kirschner MW. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol.2003;1(1):E10.
51. Tan CW, Gardiner BS, Hirokawa Y, Layton MJ, Smith DW, and Burgess AW. Wnt signalling pathway parameters for mammalian cells. PLoS One.2012;7(2): e31882.
52. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science. 1998;280(5363):596-9.
53. Nakamura T, Hamada F, Ishidate T, Anai K, Kawahara K, Toyoshima K, and Akiyama T. Axin, an inhibitor of the Wnt signalling pathway, interacts with beta-catenin, GSK-3beta and APC and reduces the beta-catenin level. Genes Cells. 1998;3(6):395-403.
54. Cliffe, A., F. Hamada, and M. Bienz, A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling. Curr Biol.2003;13(11):960-6.
55. Faux MC, Coates JL, Catimel B, Cody S, Clayton AH, Layton MJ, and Burgess AW. Recruitment of adenomatous polyposis coli and beta-catenin to axin-puncta. Oncogene.2008;27(44):5808-20.
56. Kinzler, K.W. and B. Vogelstein. Lessons from hereditary colorectal cancer. Cell.1996;87(2):159-70.
57. Furuuchi K, Tada M, Yamada H, Kataoka A, Furuuchi N, Hamada J, Takahashi M, Todo S, and Moriuchi T. Somatic mutations of the APC gene in primary breast cancers. Am J Pathol.2000;156(6):1997-2005.
58. Nathke, I.S. The adenomatous polyposis coli protein: the Achilles heel of the gut epithelium. Annu Rev Cell Dev Biol.2004;20:337-66.
59. Peifer, M. and P. Polakis. Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus. Science.2000;287(5458):1606-9.
60. Kohler EM, Derungs A, Daum G, Behrens J, and Schneikert J. Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours. Hum Mol Genet.2008;17(13):1978-87.
61. Brocardo, M., I.S. Nathke, and B.R. Henderson. Redefining the subcellular location and transport of APC:new insights using a panel of antibodies. EMBO Rep.2005;6(2):184-90.
62. Liu, X. and F. Fagotto. A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci Signal.2011;4(203): p12.
63. Zhang, F., R.L. White, and K.L. Neufeld, Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein. Mol Cell Biol.2001;21(23):8143-56.
64. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, and Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996;272(5264):1023-6.
65. Homma MK, Li D, Krebs EG, Yuasa Y, Homma Y. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein. Proc Natl Acad Sci U S A.2002;99(9):5959-64.
66. Rosin-Arbesfeld, R., F. Townsley, and M. Bienz. The APC tumour suppressor has a nuclear export function. Nature.2000;406(6799):1009-12.
67. Henderson, B.R. Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover. Nat Cell Biol.2000;2(9):653-60.
68. Neufeld KL, Zhang F, Cullen BR, and White RL. APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export. EMBO Rep.2000;1(6):519-23.
69. Cong, F. and H. Varmus. Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of beta-catenin. Proc Natl Acad Sci U S A.2004;101(9):2882-7.
70. Henderson, B.R. and F. Fagotto. The ins and outs of APC and beta-catenin nuclear transport. EMBO Rep.2002;3(9):834-9.
71. Sharma M, Jamieson C, Johnson M, Molloy MP, and Henderson BR. Specific armadillo repeat sequences facilitate beta-catenin nuclear transport in live cells via direct binding to nucleoporins Nup62, Nup153, and RanBP2/Nup358. J Biol Chem.2012;287(2):819-31.
72. Fagotto, F., U. Gluck, and B.M. Gumbiner, Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr Biol.1998;8(4):181-90.
73. Koike M, Kose S, Furuta M, Taniguchi N, Yokoya F, Yoneda Y, and Imamoto N. beta-Catenin shows an overlapping sequence requirement but distinct molecular interactions for its bidirectional passage through nuclear pores. J Biol Chem.2004;279(32):34038-47.
74. Wiechens, N. and F. Fagotto. CRM1- and Ran-independent nuclear export of beta-catenin. Curr Biol.2001;11(1):18-27.
75. Krieghoff, E., J. Behrens, and B. Mayr. Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention. J Cell Sci.2006;119(7):1453-63.
76. Roberts DM, Pronobis MI, Alexandre KM, Rogers GC, Poulton JS, Schneider DE, Jung KC, McKay DJ, and Peifer M. Defining components of the ss-catenin destruction complex and exploring its regulation and mechanisms of action during development. PLoS One.2012;7(2):e31284.
77. Sierra J, Yoshida T, Joazeiro CA, and Jones KA. The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev.2006;20(5):586-600.
78. Faux MC, Ross JL, Meeker C, Johns T, Ji H, Simpson RJ, Layton MJ, Burgess AW. Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. J Cell Sci.2004;117(3):427-39.
79. Nathke IS, Adams CL, Polakis P, Sellin JH, and Nelson WJ. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J Cell Biol.1996; 134(1):165-79.
80. Wiechens N, Heinle K, Englmeier L, Schohl A, and Fagotto F. Nucleo-cytoplasmic shuttling of Axin, a negative regulator of the Wnt-beta-catenin Pathway. J Biol Chem.2004;279(7):5263-7.
81. Salic A, Lee E, Mayer L, and Kirschner MW. Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol Cell.2000;5(3):523-32.
82. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, and Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992;256(5057):668-70.
83. Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell.2006;127(3):469-80.
84. Polakis, P. The many ways of Wnt in cancer. Curr Opin Genet Dev.2007;17(1): 45-51.
85. Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN, Vogelstein B, Kinzler KW. APC mutations occur early during colorectal tumorigenesis. Nature.1992;359(6392):235-7.
86. Xing Y, Clements WK, Kimelman D, Xu W. Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev.2003;17(22):2753-64.
87. Penman, G.A., L. Leung, and I.S. Nathke. The adenomatous polyposis coli protein (APC) exists in two distinct soluble complexes with different functions. J Cell Sci.2005;118(20):4741-50.
88. Aoki, K. and M.M. Taketo. Adenomatous polyposis coli (APC):a multi-functional tumor suppressor gene. J Cell Sci.2007;120(19):p.3327-35.
89. Heuberger, J. and W. Birchmeier. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb Perspect Biol.2010;2(2): a002915.
90. Pronobis, M.I. and M. Peifer. Wnt signaling: the many interfaces of beta-catenin. Curr Biol.2012;22(4):R137-9.
91. Louie RK, Bahmanyar S, Siemers KA, Votin V, Chang P, Stearns T, Nelson WJ, and Barth AI. Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. J Cell Sci.2004;117(7):1117-28.
92. Bahmanyar S, Kaplan DD, Deluca JG, Giddings TH Jr, O'Toole ET, Winey M, Salmon ED, Casey PJ, Nelson WJ, and Barth AI. beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes Dev.2008;22(1):91-105.
93. Alexandrova, E.M. and S.Y. Sokol. Xenopus axin-related protein: a link between its centrosomal localization and function in the Wnt/beta-catenin pathway. Dev Dyn.2010;239(1):261-70.
94. Fumoto K, Kadono M, Izumi N, and Kikuchi A. Axin localizes to the centrosome and is involved in microtubule nucleation. EMBO Rep.2009;10(6):606-13.
95. Hadjihannas, M.V., M. Bruckner, and J. Behrens. Conductin/axin2 and Wnt signalling regulates centrosome cohesion. EMBO Rep.2010;11(4):317-24.
96. Greer, Y.E. and J.S. Rubin. Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth. J Cell Biol.2011;192(6):993-1004.
97. Kose S, Imamoto N, Tachibana T, Shimamoto T, Yoneda Y. Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex. J Cell Biol.1997;39(4):841-9.
98.Shimozawa T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM, Fujita K,and Mimori-Kiyosue Y. Improving spinning disk confocal microscopy by preventingpinhole cross-talk for intravital imaging. Proc Natl Acad Sci U SA.2013;110(9):3399-404.
99. Aguila, B., M. Simaan, and S.A. Laporte. Study of G protein-coupledreceptor/beta-arrestin interactions within endosomes using FRAP. Methods MolBiol.2011;756:371-80.
100.Snapp, E.L., N. Altan, and J. Lippincott-Schwartz. Measuring protein mobility byphotobleaching GFP chimeras in living cells. Curr Protoc Cell Biol.2003;Chapter 21:p. Unit 21 1.
101.van Royen ME, Farla P, Mattern KA, Geverts B, Trapman J, and Houtsmuller AB.Fluorescence recovery after photobleaching (FRAP) to study nuclear proteindynamics in living cells. Methods Mol Biol.2009;464:363-85.
102.Staras, K., D. Mikulincer, and D. Gitler, Monitoring and quantifying dynamicphysiological processes in live neurons using Fluorescence Recovery AfterPhotobleaching. J Neurochem.2013; [Epub ahead of print].
2. Moon RT, Kohn AD, De Ferrari GV, and Kaykas A. WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet.2004;5(9):691-701.
3. Reya, T. and H. Clevers, Wnt signalling in stem cells and cancer. Nature.2005;434(7035):843-50.
4. Giles, R.H., J.H. van Es, and H. Clevers. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta.2003;653(1):1-24.
5. Polakis, P. Wnt signaling and cancer. Genes Dev.2000;14(15):1837-51.
6. Moon, R.T., J.D. Brown, and M. Torres. WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 1997; 13(4):157-62.
7. Wodarz, A. and R. Nusse. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol.1998;14:59-88.
8. Galea, M.A., A. Eleftheriou, and B.R. Henderson. ARM domain-dependent nuclear import of adenomatous polyposis coli protein is stimulated by the B56 alpha subunit of protein phosphatase 2A. J Biol Chem.2001;276(49):45833-9.
9. Neufeld KL, Nix DA, Bogerd H, Kang Y, Beckerle MC, Cullen BR, and White RL. Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. Proc Natl Acad Sci U S A.2000;97(22):12085-90.
10. Zeng L, Fagotto F, Zhang T, Hsu W, Vasicek TJ, Lee JJ, Tilghman SM, Gumbiner BM, and Costantini F. The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell.1997;90(1):181-92.
11. Kikuchi, A. Roles of Axin in the Wnt signalling pathway. Cell Signal.1999; 11(11):777-88.
12. Fiedler M, Mendoza-Topaz C, Rutherford TJ, Mieszczanek J, and Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating beta-catenin. Proc Natl Acad Sci U S A. 2011;108(5):1937-42.
13. Sakanaka, C. and L.T. Williams. Functional domains of axin. Importance of the C terminus as an oligomerization domain. J Biol Chem.1999;274(20):14090-3.
14. Huber, A.H., W.J. Nelson, and W.I. Weis, Three-dimensional structure of the armadillo repeat region of beta-catenin. Cell. 1997;90(5):871-82.
15. Huber, A.H. and W.I. Weis, The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin. Cell.2001;105(3):391-402.
16. Graham TA, Weaver C, Mao F, Kimelman D, and Xu W. Crystal structure of a beta-catenin/Tcf complex. Cell.2000; 103(6):885-96.
17. Ha NC, Tonozuka T, Stamos JL, Choi HJ, and Weis WI. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell.2004;15(4):511-21.
18. Pokutta, S. and W.I. Weis, The cytoplasmic face of cell contact sites. Curr Opin Struct Biol.2002;12(2):255-62.
19. von Kries JP, Winbeck G, Asbrand C, Schwarz-Romond T, Sochnikova N, Dell'Oro A, Behrens J, and Birchmeier W. Hot spots in beta-catenin for interactions with LEF-1, conductin and APC. Nat Struct Biol.2000;7(9):800-7.
20. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, and He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell.2002;108(6):837-47.
21. Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, and Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45:a molecular switch for the Wnt pathway. Genes Dev.2002; 16(9):1066-76.
22. Yanagawa S, Matsuda Y, Lee JS, Matsubayashi H, Sese S, Kadowaki T, and Ishimoto A. Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila. Embo J.2002;21(7):1733-42.
23. Peters JM, McKay RM, McKay JP, Graff JM. Peters JM, McKay RM, McKay JP, and Graff JM. Casein kinase I transduces Wnt signals. Nature.1999;401(6751):345-50.
24. Tickenbrock L, Kossmeier K, Rehmann H, Herrmann C, and Miiller O. Differences between the interaction of beta-catenin with non-phosphorylated and single-mimicked phosphorylated 20-amino acid residue repeats of the APC protein. J Mol Biol.2003;327(2):359-67.
25. Rubinfeld, B., D.A. Tice, and P. Polakis, Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase lepsilon. J Biol Chem.2001;276(42):39037-45.
26. Lee, E., A. Salic, and M.W. Kirschner, Physiological regulation of [beta]-catenin stability by Tcf3 and CKlepsilon. J Cell Biol.2001;154(5):983-93.
27. Gao ZH, Seeling JM, Hill V, Yochum A, and Virshup DM. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci U S A.2002;99(3):1182-7.
28. Yost C, Torres M, Miller JR, Huang E, Kimelman D, and Moon RT. The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev.1996;10(12):1443-54.
29. Dajani R, Fraser E, Roe SM, Yeo M, Good VM, Thompson V, Dale TC, Pearl LH. Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. Embo J.2003;22(3):494-501.
30. Bhanot P, Fish M, Jemison JA, Nusse R, Nathans J, and Cadigan KM. Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. Development. 1999;126(18):4175-86..
31. Willert K, Logan CY, Arora A, Fish M, and Nusse R. A Drosophila Axin homolog, Daxin, inhibits Wnt signaling. Development. 1999;126(18):4165-73.
32. Willert, K., S. Shibamoto, and R. Nusse, Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex. Genes Dev.1999; 13(14):1768-73.
33. Kobayashi M, Honma T, Matsuda Y, Suzuki Y, Narisawa R, Ajioka Y, and Asakura H. Nuclear translocation of beta-catenin in colorectal cancer. Br J Cancer.2000;82(10):1689-93.
34. Maruyama K, Ochiai A, Akimoto S, Nakamura S, Baba S, Moriya Y, and Hirohashi S. Cytoplasmic beta-catenin accumulation as a predictor of hematogenous metastasis in human colorectal cancer. Oncology.2000;59(4):302-9.
35. Munemitsu S, Albert I, Souza B, Rubinfeld B, and Polakis P. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci U S A, 1995.92(7):3046-50.
36. Fagotto F, Jho Eh, Zeng L, Kurth T, Joos T, Kaufmann C, and Costantini F. Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization. J Cell Biol.1999; 145(4):741-56.
37. Hart MJ, de los Santos R, Albert IN, Rubinfeld B, and Polakis P. Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta. Curr Biol.1998;8(10):573-81.
38. Hinoi T, Yamamoto H, Kishida M, Takada S, Kishida S, Kikuchi A. Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin. J Biol Chem.2000;275(44):34399-406.
39. Kishida S, Yamamoto H, Ikeda S, Kishida M, Sakamoto I, Koyama S, and Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem.1998;273(18):10823-6.
40. Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, Masiarz FR, Munemitsu S, Polakis P. Association of the APC gene product with beta-catenin. Science. 1993;262(5140):1731-4.
41. Su, L.K., B. Vogelstein, and K.W. Kinzler, Association of the APC tumor suppressor protein with catenins. Science. 1993;262(5140):1734-7.
42. Kimelman, D. and W. Xu, beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene.2006;25(57):7482-91.
43. Bienz, M., The subcellular destinations of APC proteins. Nat Rev Mol Cell Biol.2002;3(5):328-38.
44. Su Y, Fu C, Ishikawa S, Stella A, Kojima M, Shitoh K, Schreiber EM, Day BW, and Liu B. APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase. Mol Cell.2008;32(5):652-61.
45. Li VS, Ng SS, Boersema PJ, Low TY, Karthaus WR, Gerlach JP, Mohammed S, Heck AJ, Maurice MM, Mahmoudi T, and Clevers H. Wnt signaling through inhibition of beta-catenin degradation in an intact Axinl complex. Cell.2012;149(6):1245-56.
46. Roberts DM, Pronobis MI, Poulton JS, Waldmann JD, Stephenson EM, Hanna S, and Peifer M. Deconstructing the sscatenin destruction complex:mechanistic roles for the tumor suppressor APC in regulating Wnt signaling. Mol Biol Cell.2011;22(11):845-63.
47. Faux MC, Coates JL, Kershaw NJ, Layton MJ, and Burgess AW. Independent interactions of phosphorylated beta-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions. PLoS One. 5(11):e14127.
48. Stambolic, V., L. Ruel, and J.R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol.1996;6(12):1664-8.
49. Taelman VF, Dobrowolski R, Plouhinec JL, Fuentealba LC, Vorwald PP, Gumper I, Sabatini DD, and De Robertis EM. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell.2010;143(7):1136-48.
50. Lee E, Salic A, Kruger R, Heinrich R, and Kirschner MW. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol.2003;1(1):E10.
51. Tan CW, Gardiner BS, Hirokawa Y, Layton MJ, Smith DW, and Burgess AW. Wnt signalling pathway parameters for mammalian cells. PLoS One.2012;7(2): e31882.
52. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Wirtz R, Kuhl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science. 1998;280(5363):596-9.
53. Nakamura T, Hamada F, Ishidate T, Anai K, Kawahara K, Toyoshima K, and Akiyama T. Axin, an inhibitor of the Wnt signalling pathway, interacts with beta-catenin, GSK-3beta and APC and reduces the beta-catenin level. Genes Cells. 1998;3(6):395-403.
54. Cliffe, A., F. Hamada, and M. Bienz, A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling. Curr Biol.2003;13(11):960-6.
55. Faux MC, Coates JL, Catimel B, Cody S, Clayton AH, Layton MJ, and Burgess AW. Recruitment of adenomatous polyposis coli and beta-catenin to axin-puncta. Oncogene.2008;27(44):5808-20.
56. Kinzler, K.W. and B. Vogelstein. Lessons from hereditary colorectal cancer. Cell.1996;87(2):159-70.
57. Furuuchi K, Tada M, Yamada H, Kataoka A, Furuuchi N, Hamada J, Takahashi M, Todo S, and Moriuchi T. Somatic mutations of the APC gene in primary breast cancers. Am J Pathol.2000;156(6):1997-2005.
58. Nathke, I.S. The adenomatous polyposis coli protein: the Achilles heel of the gut epithelium. Annu Rev Cell Dev Biol.2004;20:337-66.
59. Peifer, M. and P. Polakis. Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus. Science.2000;287(5458):1606-9.
60. Kohler EM, Derungs A, Daum G, Behrens J, and Schneikert J. Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours. Hum Mol Genet.2008;17(13):1978-87.
61. Brocardo, M., I.S. Nathke, and B.R. Henderson. Redefining the subcellular location and transport of APC:new insights using a panel of antibodies. EMBO Rep.2005;6(2):184-90.
62. Liu, X. and F. Fagotto. A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci Signal.2011;4(203): p12.
63. Zhang, F., R.L. White, and K.L. Neufeld, Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein. Mol Cell Biol.2001;21(23):8143-56.
64. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, and Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996;272(5264):1023-6.
65. Homma MK, Li D, Krebs EG, Yuasa Y, Homma Y. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein. Proc Natl Acad Sci U S A.2002;99(9):5959-64.
66. Rosin-Arbesfeld, R., F. Townsley, and M. Bienz. The APC tumour suppressor has a nuclear export function. Nature.2000;406(6799):1009-12.
67. Henderson, B.R. Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover. Nat Cell Biol.2000;2(9):653-60.
68. Neufeld KL, Zhang F, Cullen BR, and White RL. APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export. EMBO Rep.2000;1(6):519-23.
69. Cong, F. and H. Varmus. Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of beta-catenin. Proc Natl Acad Sci U S A.2004;101(9):2882-7.
70. Henderson, B.R. and F. Fagotto. The ins and outs of APC and beta-catenin nuclear transport. EMBO Rep.2002;3(9):834-9.
71. Sharma M, Jamieson C, Johnson M, Molloy MP, and Henderson BR. Specific armadillo repeat sequences facilitate beta-catenin nuclear transport in live cells via direct binding to nucleoporins Nup62, Nup153, and RanBP2/Nup358. J Biol Chem.2012;287(2):819-31.
72. Fagotto, F., U. Gluck, and B.M. Gumbiner, Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr Biol.1998;8(4):181-90.
73. Koike M, Kose S, Furuta M, Taniguchi N, Yokoya F, Yoneda Y, and Imamoto N. beta-Catenin shows an overlapping sequence requirement but distinct molecular interactions for its bidirectional passage through nuclear pores. J Biol Chem.2004;279(32):34038-47.
74. Wiechens, N. and F. Fagotto. CRM1- and Ran-independent nuclear export of beta-catenin. Curr Biol.2001;11(1):18-27.
75. Krieghoff, E., J. Behrens, and B. Mayr. Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention. J Cell Sci.2006;119(7):1453-63.
76. Roberts DM, Pronobis MI, Alexandre KM, Rogers GC, Poulton JS, Schneider DE, Jung KC, McKay DJ, and Peifer M. Defining components of the ss-catenin destruction complex and exploring its regulation and mechanisms of action during development. PLoS One.2012;7(2):e31284.
77. Sierra J, Yoshida T, Joazeiro CA, and Jones KA. The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev.2006;20(5):586-600.
78. Faux MC, Ross JL, Meeker C, Johns T, Ji H, Simpson RJ, Layton MJ, Burgess AW. Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. J Cell Sci.2004;117(3):427-39.
79. Nathke IS, Adams CL, Polakis P, Sellin JH, and Nelson WJ. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J Cell Biol.1996; 134(1):165-79.
80. Wiechens N, Heinle K, Englmeier L, Schohl A, and Fagotto F. Nucleo-cytoplasmic shuttling of Axin, a negative regulator of the Wnt-beta-catenin Pathway. J Biol Chem.2004;279(7):5263-7.
81. Salic A, Lee E, Mayer L, and Kirschner MW. Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol Cell.2000;5(3):523-32.
82. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, and Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992;256(5057):668-70.
83. Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell.2006;127(3):469-80.
84. Polakis, P. The many ways of Wnt in cancer. Curr Opin Genet Dev.2007;17(1): 45-51.
85. Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN, Vogelstein B, Kinzler KW. APC mutations occur early during colorectal tumorigenesis. Nature.1992;359(6392):235-7.
86. Xing Y, Clements WK, Kimelman D, Xu W. Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev.2003;17(22):2753-64.
87. Penman, G.A., L. Leung, and I.S. Nathke. The adenomatous polyposis coli protein (APC) exists in two distinct soluble complexes with different functions. J Cell Sci.2005;118(20):4741-50.
88. Aoki, K. and M.M. Taketo. Adenomatous polyposis coli (APC):a multi-functional tumor suppressor gene. J Cell Sci.2007;120(19):p.3327-35.
89. Heuberger, J. and W. Birchmeier. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb Perspect Biol.2010;2(2): a002915.
90. Pronobis, M.I. and M. Peifer. Wnt signaling: the many interfaces of beta-catenin. Curr Biol.2012;22(4):R137-9.
91. Louie RK, Bahmanyar S, Siemers KA, Votin V, Chang P, Stearns T, Nelson WJ, and Barth AI. Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. J Cell Sci.2004;117(7):1117-28.
92. Bahmanyar S, Kaplan DD, Deluca JG, Giddings TH Jr, O'Toole ET, Winey M, Salmon ED, Casey PJ, Nelson WJ, and Barth AI. beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes Dev.2008;22(1):91-105.
93. Alexandrova, E.M. and S.Y. Sokol. Xenopus axin-related protein: a link between its centrosomal localization and function in the Wnt/beta-catenin pathway. Dev Dyn.2010;239(1):261-70.
94. Fumoto K, Kadono M, Izumi N, and Kikuchi A. Axin localizes to the centrosome and is involved in microtubule nucleation. EMBO Rep.2009;10(6):606-13.
95. Hadjihannas, M.V., M. Bruckner, and J. Behrens. Conductin/axin2 and Wnt signalling regulates centrosome cohesion. EMBO Rep.2010;11(4):317-24.
96. Greer, Y.E. and J.S. Rubin. Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth. J Cell Biol.2011;192(6):993-1004.
97. Kose S, Imamoto N, Tachibana T, Shimamoto T, Yoneda Y. Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex. J Cell Biol.1997;39(4):841-9.
98.Shimozawa T, Yamagata K, Kondo T, Hayashi S, Shitamukai A, Konno D,Matsuzaki F, Takayama J, Onami S, Nakayama H, Kosugi Y, Watanabe TM, Fujita K,and Mimori-Kiyosue Y. Improving spinning disk confocal microscopy by preventingpinhole cross-talk for intravital imaging. Proc Natl Acad Sci U SA.2013;110(9):3399-404.
99. Aguila, B., M. Simaan, and S.A. Laporte. Study of G protein-coupledreceptor/beta-arrestin interactions within endosomes using FRAP. Methods MolBiol.2011;756:371-80.
100.Snapp, E.L., N. Altan, and J. Lippincott-Schwartz. Measuring protein mobility byphotobleaching GFP chimeras in living cells. Curr Protoc Cell Biol.2003;Chapter 21:p. Unit 21 1.
101.van Royen ME, Farla P, Mattern KA, Geverts B, Trapman J, and Houtsmuller AB.Fluorescence recovery after photobleaching (FRAP) to study nuclear proteindynamics in living cells. Methods Mol Biol.2009;464:363-85.
102.Staras, K., D. Mikulincer, and D. Gitler, Monitoring and quantifying dynamicphysiological processes in live neurons using Fluorescence Recovery AfterPhotobleaching. J Neurochem.2013; [Epub ahead of print].