Park Mi Kyung, Lee Hye Ja, Shin Jennifer, Noh Minsoo, Kim Soo Youl, Lee Chang Hoon
College of Pharmacy, Dongguk University, Seoul, Korea.
Biochim Biophys Acta. 2011 Dec;1811(12):1021-9. doi: 10.1016/j.bbalip.2011.07.007. Epub 2011 Jul 26.
Sphingosylphosphorylcholine (SPC) is found at increased levels in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. In this study, we investigated the role and molecular mechanisms of Tgase-2 in SPC-induced K8 phosphorylation and perinuclear reorganization in PANC-1 cells (PAN(WT)), and in PANC-1 cells that stably expressed shTgase-2 or Tgase-2 (PAN(shTg2) and PAN(Tg2)). SPC induces the expression of Tgase-2 in a time- and dose-dependent manner. Gene silencing of Tgase-2 or cystamine suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8 and suppressed the SPC-induced migration of PANC-1 cells. An inhibitor of c-Jun N-terminal kinase (JNK), SP600125, suppressed the SPC-induced phosphorylation of serine 431 in K8 and keratin reorganization. Next, we examined the effect of Tgase-2 on JNK activation of serine 431 phosphorylation in K8. Tgase-2 gene silencing suppressed the expression of active form JNK (pJNK). Constitutive or tetracyclin-induced conditional expression of Tgase-2 increased the levels of pJNK. Tgase-2 was coimmunoprecipitated with K8 and JNK. In addition, K8 was coimmunoprecipitated with Tgase-2 and JNK. JNK was also coimmunoprecipitated with K8 and Tgase-2. Overall, these results suggest that Tgase-2 is involved in SPC-induced phosphorylation and perinuclear reorganization of K8 by activating JNK and forming a triple complex with K8 and JNK. Therefore, SPC-induced Tgase-2 might be a new target for modulating keratin reorganization, metastasis of cancer cells and JNK activation.
鞘氨醇磷酸胆碱（SPC）在肿瘤患者的恶性腹水中水平升高，并诱导角蛋白8（K8）丝的核周重排，这有助于转移癌细胞的粘弹性。在本研究中，我们研究了转谷氨酰胺酶2（Tgase-2）在SPC诱导的PANC-1细胞（PAN(WT)）以及稳定表达shTgase-2或Tgase-2的PANC-1细胞（PAN(shTg2)和PAN(Tg2)）中K8磷酸化和核周重排中的作用及分子机制。SPC以时间和剂量依赖性方式诱导Tgase-2的表达。Tgase-2或胱胺的基因沉默抑制了SPC诱导的K8磷酸化和核周重排，并抑制了SPC诱导的PANC-1细胞迁移。c-Jun氨基末端激酶（JNK）抑制剂SP600125抑制了SPC诱导的K8中丝氨酸431的磷酸化和角蛋白重排。接下来，我们研究了Tgase-2对K8中丝氨酸431磷酸化的JNK激活的影响。Tgase-2基因沉默抑制了活性形式JNK（pJNK）的表达。Tgase-2的组成型或四环素诱导的条件性表达增加了pJNK的水平。Tgase-2与K8和JNK共免疫沉淀。此外，K8与Tgase-2和JNK共免疫沉淀。JNK也与K8和Tgase-2共免疫沉淀。总体而言，这些结果表明Tgase-2通过激活JNK并与K8和JNK形成三聚体复合物参与SPC诱导的K8磷酸化和核周重排。因此，SPC诱导的Tgase-2可能是调节角蛋白重排、癌细胞转移和JNK激活的新靶点。
