Roczniak-Ferguson Agnes, Reynolds Albert B
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
J Cell Sci. 2003 Oct 15;116(Pt 20):4201-12. doi: 10.1242/jcs.00724. Epub 2003 Sep 2.
P120-catenin is the prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins involved in cell-cell adhesion. Interestingly, all members of the p120 subfamily have also been observed in the nucleus, suggesting that they have additional roles that have yet to be determined. Here, we have developed a novel model system for studying the nucleocytoplasmic shuttling capabilities of p120. We show that simultaneous deletion of both of the conventional nuclear localization sequences (NLSs) in p120 had little effect on its nuclear localization. Instead, the Armadillo repeat domain was essential, and deletion of Arm repeat 3 or Arm repeat 5 eliminated nuclear entry despite the presence of both NLSs. In addition, deletion of Arm repeat 8 resulted in constitutive nuclear localization of p120-3A in both E-cadherin-positive and -negative cell lines. Thus, the core shuttling functions are dependent on the Arm domain. We have also identified two regions within the N-terminus of p120 that modulate nuclear shuttling dynamics of p120. In cadherin-deficient cells, normal epithelial morphology could be restored by both WT-E-cadherin and p120 uncoupled E-cadherin mutants, but only WT-E-cadherin strongly reduced nuclear localization of p120. Moreover, structural changes in p120 that reduced its affinity for E-cadherin increased p120 nuclear localization. Thus, reduced shuttling in the presence of E-cadherin is principally due to sequestration, a condition that is probably dynamic under normal circumstances but completely lost in metastatic cells that have downregulated E-cadherin. Notably, Arm repeats 3 and 5 are necessary for both E-cadherin binding and nuclear translocation, indicating that these repeats have dual roles. Surprisingly, in the absence of E-cadherin there was significant colocalization of cytoplasmic p120 with elements of the tubulin cytoskeleton, particularly in perinuclear locations. Depolymerizing microtubules with nocodazole increased nuclear p120, whereas stabilizing tubulin with taxol reduced nuclear p120 and strongly increased p120 association with microtubules. Thus, p120 has intrinsic nucleocytoplasmic shuttling activity that is modulated, in part, by extrinsic factors such as cadherin binding and interactions with the microtubule network.
P120连环蛋白是犰狳重复结构域（Arm结构域）蛋白亚家族的典型成员，参与细胞间黏附。有趣的是，p120亚家族的所有成员也在细胞核中被观察到，这表明它们还有尚未确定的其他作用。在此，我们开发了一种新型模型系统来研究p120的核质穿梭能力。我们发现，同时缺失p120中两个传统的核定位序列（NLSs）对其核定位影响很小。相反，犰狳重复结构域至关重要，缺失Arm重复序列3或Arm重复序列5会消除核进入，尽管存在两个NLSs。此外，缺失Arm重复序列8导致p120 - 3A在E - 钙黏蛋白阳性和阴性细胞系中均出现组成型核定位。因此，核心穿梭功能依赖于Arm结构域。我们还在p120的N端鉴定出两个区域，它们调节p120的核穿梭动力学。在钙黏蛋白缺陷细胞中，野生型E - 钙黏蛋白和p120非偶联E - 钙黏蛋白突变体均可恢复正常上皮形态，但只有野生型E - 钙黏蛋白能强烈降低p120的核定位。此外，降低p120与E - 钙黏蛋白亲和力的结构变化会增加p120的核定位。因此，在E - 钙黏蛋白存在下穿梭减少主要是由于隔离，这种情况在正常情况下可能是动态的，但在下调E - 钙黏蛋白的转移细胞中则完全丧失。值得注意的是，Arm重复序列3和5对于E - 钙黏蛋白结合和核转位都是必需的，这表明这些重复序列具有双重作用。令人惊讶的是，在没有E - 钙黏蛋白的情况下，细胞质p120与微管细胞骨架成分有显著共定位，特别是在核周位置。用诺考达唑解聚微管会增加核内p120，而用紫杉醇稳定微管蛋白会减少核内p120并强烈增加p120与微管的结合。因此，p120具有内在的核质穿梭活性，部分受外在因素如钙黏蛋白结合和与微管网络相互作用的调节。
