Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, NV 89135, USA.
Neurochem Int. 2010 Mar;56(4):546-53. doi: 10.1016/j.neuint.2009.12.017. Epub 2010 Jan 4.
Previous studies from our laboratory have shown that the cGMP/protein kinase G (PKG) signaling pathway plays an essential role in preventing spontaneous apoptosis in neural cells; however, the mechanism is not understood. A potential downstream target of PKG is the apoptosis-regulating protein Bad, which contains a sequence around its serine 155 (ser155 in mouse Bad, equivalent to ser118 in human Bad) predicted to be a consensus motif for PKG-catalyzed phosphorylation. Using both in vitro and cell-based experiments, we determined if PKG phosphorylates Bad at ser155 and if blocking/stimulating PKG-catalyzed Bad phosphorylation causes pro-apoptotic/anti-apoptotic responses. Recombinant PKG type-Ialpha (PKG-Ialpha) was found to directly phosphorylate recombinant Bad at ser155 in vitro. In N1E-115 mouse neural cells, which naturally express PKG-Ialpha as the predominant PKG isoform, addition of 8-Br-cGMP (0.1-1.0 mM), a cell-permeable direct PKG-Ialpha activator, increased ser155 phosphorylation of Bad. ODQ (50 microM), a soluble guanylyl cyclase inhibitor that lowers cGMP/PKG activity, decreased serum-induced ser155 phosphorylation of Bad and induced apoptosis in N1E-115 cells. Treatment with DT-2 and DT-3, selective PKG-Ialpha inhibitors, both decreased Bad ser155 phosphorylation and induced apoptosis. The data indicate that PKG-Ialpha directly phosphorylates Bad at ser155, which may participate in cGMP/PKG-induced anti-apoptotic/cytoprotective effects in neural cells.
先前我们实验室的研究表明，cGMP/蛋白激酶 G（PKG）信号通路在防止神经细胞自发性凋亡中起着至关重要的作用；然而，其机制尚不清楚。PKG 的一个潜在下游靶标是凋亡调节蛋白 Bad，其丝氨酸 155 周围（在小鼠 Bad 中为 ser155，相当于人 Bad 中的 ser118）存在一个序列，预测其为 PKG 催化磷酸化的共有基序。我们使用体外和基于细胞的实验来确定 PKG 是否在 ser155 处磷酸化 Bad，以及阻断/刺激 PKG 催化的 Bad 磷酸化是否会引起促凋亡/抗凋亡反应。重组 PKG I 型α（PKG-Ialpha）被发现可在体外直接将重组 Bad 磷酸化至 ser155。在自然表达 PKG-Ialpha 作为主要 PKG 同工型的 N1E-115 小鼠神经细胞中，添加 8-Br-cGMP（0.1-1.0mM），一种细胞可渗透的直接 PKG-Ialpha 激活剂，可增加 Bad 的 ser155 磷酸化。ODQ（50μM），一种可溶性鸟苷酸环化酶抑制剂，可降低 cGMP/PKG 活性，可降低血清诱导的 Bad 的 ser155 磷酸化并诱导 N1E-115 细胞凋亡。用 DT-2 和 DT-3 处理，选择性 PKG-Ialpha 抑制剂，均可降低 Bad 的 ser155 磷酸化并诱导细胞凋亡。数据表明 PKG-Ialpha 可直接将 Bad 磷酸化至 ser155，这可能参与 cGMP/PKG 诱导的神经细胞抗凋亡/细胞保护作用。
