PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity.
PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity.
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DOI：
10.1242/jcs.120154
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发表时间：
2014-02-01
影响因子：
4
通讯作者：
Sastry SK
中科院分区：
文献类型：
--
作者：
Espejo R;Jeng Y;Paulucci-Holthauzen A;Rengifo-Cam W;Honkus K;Anastasiadis PZ;Sastry SK
酪氨酸磷酸化与粘附体连接蛋白p120连环蛋白（p120）的调节有关，但其机制尚不明确。在这里，我们使用底物捕获技术证明，p120是上皮细胞中蛋白酪氨酸磷酸酶PTP-PEST的直接靶标。在结肠癌细胞中，稳定的shRNA敲除PTP-PEST导致p120细胞质池增加，同时酪氨酸磷酸化增强，与e -钙粘蛋白的关联降低。与此一致的是，PTP-PEST敲除细胞在胶原底物上表现出增加的运动，增强的Rac1和降低的RhoA活性。此外，p120的定位在肌动蛋白丰富的突起和板足处增强，并与鸟嘌呤核苷酸交换因子、VAV2和接触增加相关。VAV2的交换因子活性通过PTP-PEST敲除而增强，而VAV2 c端结构域或DH结构域突变体的过表达会阻断细胞运动。点突变分析发现p120 n端区域的酪氨酸335是PTP-PEST去磷酸化位点。p120的Y335F突变体不能诱导“p120表型”，不能与VAV2相互作用，不能刺激细胞运动或激活Rac1。综上所述，这些数据表明PTP-PEST通过控制p120的分布和磷酸化及其控制Rho GTPase活性的有效性来影响上皮细胞的运动。
Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the ‘p120 phenotype’, interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity.