Department of Internal Medicine, University of Manitoba, 843 JBRC/715 McDermot Avenue, Winnipeg, MB R3E 3P4, Canada.
BMC Cancer. 2012 Jul 3;12:277. doi: 10.1186/1471-2407-12-277.
Transglutaminase 2 (TG2) and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB), protein kinase B (PKB/Akt) and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA) induced phosphorylation of TG2 at serine-216 (Ser(216)) regulates TG2 function and facilitates protein-protein interaction. However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored.
In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)) overexpressing native TG2 or mutant-TG2 (m-TG2) lacking Ser(216) phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP) stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS) analysis and cell migration assay respectively.
PKA activation in TG2 overexpressing MEF(tg2-/-) cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEF(tg2-/-) cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells overexpressing TG2 and m-TG2 further supporting the role of TG2 phosphorylation in NF-κB activation and in the downregulation of PTEN.
Collectively, these data suggest that phosphorylation of TG2 at Ser(216) plays a role in TG2 mediated activation of NF-κB, Akt and in the downregulation of PTEN. Blocking TG2 phosphorylation may provide a novel strategy to attenuate NF-κB activation and downregulation of PTEN in TG2 overexpressing cancers.
转谷氨酰胺酶 2（TG2）及其磷酸化在许多癌细胞类型中一直被发现上调。在分子水平上，TG2 与核因子-κB（NF-κB）、蛋白激酶 B（PKB/Akt）的激活有关，与磷酸酶和张力蛋白同源物缺失 10 号染色体（PTEN）的下调有关。然而，其中涉及的潜在机制尚不清楚。我们已经报道，蛋白激酶 A（PKA）诱导 TG2 丝氨酸-216（Ser（216））的磷酸化调节 TG2 功能并促进蛋白质-蛋白质相互作用。然而，TG2 磷酸化在调节 NF-κB、Akt 和 PTEN 中的作用尚未得到探索。
在这项研究中，我们使用胚胎成纤维细胞（MEF（tg2-/-））过表达天然 TG2 或缺乏 Ser（216）磷酸化位点的突变型-TG2（m-TG2），研究了 TG2 磷酸化对 NF-κB、Akt 和 PTEN 的影响，同时用二丁酰环 AMP（db-cAMP）刺激。通过荧光激活细胞分选（FACS）分析和细胞迁移测定分别确定细胞周期和细胞迁移的功能后果。
与空载体转染对照细胞相比，PKA 激活在过表达 TG2 的 MEF（tg2-/-）细胞中导致 NF-κB 激活和 Akt 磷酸化增加，这分别通过报告基因测定和免疫印迹分析确定。在过表达 m-TG2 的 MEF（tg2-/-）细胞中未观察到这些效应。同样，与空载体对照和 m-TG2 转染细胞相比，在过表达 TG2 的细胞中发现 PTEN 的 mRNA 和蛋白水平均显著下调。此外，Akt 的激活与 NF-κB 的同时激活和 PTEN 的减少相关，表明 TG2 对 Akt 激活的促进作用发生在 PTEN 依赖性方式中。在过表达 TG2 和 m-TG2 的 MCF-7 和 T-47D 乳腺癌细胞中也得到了类似的结果，进一步支持了 TG2 磷酸化在 NF-κB 激活和 PTEN 下调中的作用。
总之，这些数据表明，TG2 丝氨酸-216 （Ser（216））的磷酸化在 TG2 介导的 NF-κB、Akt 激活和 PTEN 下调中发挥作用。阻断 TG2 磷酸化可能为在 TG2 过表达的癌症中减弱 NF-κB 激活和下调 PTEN 提供一种新的策略。
