探討IKK調控PMA誘導c-fos基因表現之機轉

Abstract

PMA 為一已知的蛋白磷酸激酶C (PKC) 活化劑, 可活化轉錄因子NF-kappaB及c-Fos蛋白表現。我們欲探討此兩條活化路徑的關係，因此比較腫瘤壞死因子-alpha (TNF-alpha) 和PMA在不同種細胞的作用。首先，我們發現PMA所誘導的p65蛋白絲胺酸 (Serine) 536磷酸化需要IKKalpha及IKKbeta，而TNF-alpha的作用則非如此。除此之外，在多種細胞我們發現TNF-alpha 會引起強烈IKKalpha/beta 活性區的絲胺酸176及絲胺酸177磷酸化，相反的，PMA偏好於IKKalpha/beta活性區的絲胺酸180及絲胺酸181產生磷酸化，並且此磷酸化作用與PKC-MEK-ERK路徑有關，但與RSK及MSK無關。然而，此種磷酸化作用並未參與PMA所誘導的IkappaBalpha蛋白降解、p65蛋白絲胺酸536磷酸化、p65蛋白進入細胞核以及轉錄因子NF-kappaB的活化。出乎意料，我們發現PMA所誘導的c-Fos蛋白表現需要IKKalpha/IKKbeta/IKKgamma複合物，而此種相依性也在p65蛋白缺陷的老鼠胚胎纖維母細胞 (MEF) 中發現。更進一步，我們在老鼠c-fos基因啟動子 (promoter) 區域中找到了一個可能被轉錄因子NF-kappaB所結合的區域。經由實驗證明，在PMA的刺激之下，p65蛋白同質雙聚體 (p65 homodimer) 可以和CBP/p300蛋白結合到此區域進行基因調控，而此種調控與TCF-SRF-SRE複合物相關之調控無關。簡而言之，此篇研究發現IKKalpha/beta蛋白磷酸化的新機制，也證實PMA所誘導的c-fos基因調控需要IKKalpha/beta蛋白參與，而c-fos基因啟動子之轉錄因子NF-kappaB結合位置的發現，將可對於轉錄因子NF-kappaB及轉錄因子AP-1兩者之間的關係，提供一個新的思考點及研究方針。

Phorbol 12-myristate 13-acetate (PMA) is a PKC activator known for triggering NF-kappaB activation and c-fos expression. To further understand the functional link of both events, we compared the actions of TNF-alpha and PMA in various cell types. We found that PMA- but not TNF-alpha-induced p65 Ser536 phosphorylation in mouse embryonic fibroblast (MEF) is IKKalpha/beta dependent. Additionally, compared to TNF-alpha, which induced dramatically IKKalpha/beta phosphorylation at Ser176/177, PMA preferentially induced IKKalpha/beta phosphorylation at Ser180/181 within its active loop, and this phosphorylation of IKKalpha/beta by PMA is through a novel mechanism depending on PKC-MEK-ERK axis but not RSK or MSK. Moreover, this phosphorylation does not contribute to PMA-induced IkappaBalpha degradation, p65 Ser536 phosphorylation, p65 nuclear translocation and NF-kappaB activation. Surprisingly, we found that PMA-induced immediately early gene c-fos expression is suppressed in IKKalpha-/-, IKKbeta-/-, IKKgamma-/- and p65-/- MEF, suggesting the involvement of NF-kappaB in c-fos gene transcription. Furthermore, we indentified a previously uncharacterized NF-kappaB binding site located in the mouse c-fos promoter. Under PMA stimulation, p65 homodimer in association with CBP/p300 is capable of binding to this site, and this action is independent of TCF-SRF-SRE ternary complex regulation. In summary, this study provides different views of IKK phosphorylation mechanism. Importantly, the requirement of IKK complex in PMA-induced c-fos expression and the NF-kappaB binding site found in c-fos promoter shed light on the crosstalk between NF-kappaB and AP-1 transcription factors.