表皮生長因子受體訊息路徑調控 CTEN 磷酸化之功能與機制探討

Abstract

表皮生長因子受體 (EGFR) 訊息路徑可以藉由 MAPK、PI3K、STAT 等路徑調控細胞的基因表現進而影響細胞的生理機制，tensin 家族的蛋白質為 EGFR 路徑調控的下游分子。C-terminal tensin like (CTEN) 屬於 tensin 家族一員，也為集中附著點 (focal adhesion) 蛋白質，它在細胞的附著、移動與腫瘤發生的過程中扮演重要的角色。本實驗室先前的研究中發現 CTEN 在大腸癌細胞中有受到磷酸化的現象，也發現了 T347、S350、S386 三個胺基酸位點會受到 EGFR 訊息路徑的活化而磷酸化，並進一步證實是藉由 MAPK 與 PI3K 訊息路徑調控 CTEN 上此三個位點的磷酸化。本論文想要進一步探討 T347、S350、S386 三個位點的磷酸化對於細胞生理現象的影響，因此建構了能穩定表現野生型 CTEN (CTEN-WT) 與無法被磷酸化的突變 CTEN (CTEN-M3) 的 HeLa 細胞株與 293A 細胞株，並測試細胞的增生與移動能力，實驗結果顯示，沒有磷酸化的CTEN 並不影響受 EGFR 訊息路徑的活化的細胞增生與移動能力，表示在 HeLa 與 293A 細胞中此三個位點的磷酸化 CTEN 並不參與細胞增生與移動能力的調控。另一方面，我們也進行 T347、S350、S386 受激酶磷酸化的機制研究，但實驗中 AKT 與 ERK 激酶並無法將包含 T347、S350、S386 的 CTEN 230-437 片段磷酸化。CTEN 此三個位點的磷酸化是否需要 CTEN 其餘片段的輔助，與此三個位點是否為 AKT 或 ERK 激酶進行磷酸化的基質仍有待進一步的探討。

Epithelial growth factor receptor (EGFR) signaling pathway regulates multiple gene expression and cell behaviors through MAPK, PI3K or STAT pathways. Tensin family is one of the downstream substrates regulated by EGFR signaling. CTEN is a member of tensin family and a focal adhesion protein. CTEN also participates in regulation of cell adhesion, cell migration, and tumorigenesis. In our previous research, we found that CTEN is phosphorylated in both cytoplasm and nucleus in colon cancer cell, and three amino acid residues, T347, S350 and S386, were identified to be phosphorylated upon EGFR signaling acitivation. We also proved that EGFR signaling pathway regulates the phosphorylation of T347, S350 and S386 through MAPK and PI3K pathways. In this study, we want to further investigate the impact of CTEN phosphorylation on cell behaviors. Therefore, we first established stable clones that express wild type CTEN (CTEN-WT) and mutant CTEN (CTEN-M3) with mutated phosphorylation sites in HeLa and 293A cell lines. The EGFR-activated cell proliferation and migration abilities of CTEN-M3 cells showed no significant difference comparing to those of CTEN-WT cells. It suggests that the phosphorylation of T347, S350, S386 on CTEN does not participate in the regulation of cell migration and proliferation when EGFR signaling is activated. On the other hand, we also investigated the phosphorylation mechanism of T347, S350, S386 sites by kinases. However, the CTEN 230-437 fragment containing T347, S350 and S386 was not phosphorylated in this experiment. Whether the phosphorylation of these three sites needs the other part of CTEN to assist and whether they are the substrates of AKT or ERK kinases warrant further investigation.