Tatsumi Yasuaki, Cho Yong-Yeon, He Zhiwei, Mizuno Hideya, Seok Choi Hong, Bode Ann M, Dong Zigang
Hormel Institute, University of Minnesota, Austin, Minnesota.
Cancer Res. 2006 Jun 1;66(11):5968-74. doi: 10.1158/0008-5472.CAN-05-4664.
Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)-dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser(63) and Ser(73). Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability.
桩蛋白是Src酪氨酸癌基因激酶的底物，参与细胞转化、细胞铺展、迁移以及由丝裂原活化蛋白激酶信号级联介导的癌症发展过程。在此，我们表明桩蛋白在表皮生长因子（EGF）或12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）诱导的皮肤细胞转化中起关键作用。为了研究桩蛋白在细胞转化中作用的机制，我们通过导入小干扰RNA - 桩蛋白（si - 桩蛋白）建立了稳定转染的桩蛋白敲低细胞系。与si - 对照细胞相比，si - 桩蛋白细胞显示出由EGF或TPA诱导的细胞增殖和非锚定依赖性细胞转化受到显著抑制。在si - 桩蛋白细胞中，激活蛋白 - 1（AP - 1）依赖性荧光素酶活性降低与AP - 1 DNA结合活性受到抑制相对应。重要的是，桩蛋白的敲低抑制了EGF或TPA诱导的c - Jun在Ser(63)和Ser(73)位点的磷酸化。此外，si - 桩蛋白细胞中c - Jun总蛋白水平显著降低，且依赖于血清剥夺时间。用蛋白酶体抑制剂乳胞素处理可使si - 桩蛋白细胞中c - Jun的下调得到恢复，但用溶酶体抑制剂亮抑蛋白酶肽处理则不能。这些结果清楚地证明了桩蛋白调节c - Jun蛋白水平，并最有可能通过调节c - Jun的稳定性在细胞转化中起关键作用。
