Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine, Molecular Biology Institute, University of California, Los Angeles, California 90095-1786, USA.
J Cell Physiol. 2010 May;223(2):309-16. doi: 10.1002/jcp.22036.
Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.
神经降压素（NT）刺激蛋白激酶 D1（PKD1）、细胞外信号调节激酶（ERK）、c-Jun N 末端激酶（JNK）和人胰腺腺癌细胞系 PANC-1 的 DNA 合成。为了确定 PKD1 过表达对这些生物学反应的影响，我们生成了诱导稳定的 PANC-1 克隆，这些克隆可以响应蜕皮激素类似物 ponasterone-A（PonA）表达野生型（WT）或激酶失活（K618N）形式的 PKD1。NT 强烈刺激 WT 和 PANC-1 细胞的克隆衍生物中的 c-Jun Ser（63）磷酸化。PonA 诱导的 WT PKD1 表达，但不是 K618N PKD1 表达，快速阻断 NT 介导的 c-Jun Ser（63）磷酸化，无论是在 MKK4 水平还是在其上游，MKK4 是一种导致 JNK 激活的双特异性激酶。这是第一个证明 PKD1 抑制 PANC-1 细胞中 NT 诱导的 JNK/cJun 激活的实验。相比之下，PKD1 过表达显著增加了这些细胞中 NT 诱导的 ERK 激活的持续时间。PKD1 信号对促有丝分裂 ERK 和促凋亡 JNK/c-Jun 途径的相互影响促使我们检查 PKD1 过表达是否促进 PANC-1 细胞的 DNA 合成和增殖。我们的结果表明，PKD1 过表达增加了在普通培养皿或聚羟乙基甲基丙烯酸酯[Poly-（HEMA）]涂层培养皿中培养的 PANC-1 细胞的 DNA 合成和细胞数量，以消除细胞黏附（非锚定依赖性生长）。此外，PKD1 过表达显著增强了 NT（1-10 nM）诱导的 DNA 合成。这些结果表明，PKD1 介导了 PANC-1 中的有丝分裂信号，并表明该酶可能成为开发限制这些细胞增殖的治疗药物的新靶点。
