Regulation of MLK3 by oxidative stress in colon cancer cells

 描述(申请人提供)：混合谱系激酶3(MLK3)是一种丝氨酸/苏氨酸丝裂原活化蛋白激酶(MAP3K)，调节多个MAPK信号通路，以响应有丝分裂和应激刺激。根据细胞环境的不同，MLK3可以磷酸化MKK4或MKK7 MAPK激酶(MAP2K)，激活c-Jun氨基末端激酶(JNK)，从而触发细胞死亡。另外，MLK3通过一种不依赖于激酶的支架功能，可以激活B-Raf和细胞外信号调节激酶(ERK)信号，从而促进细胞增殖。MLK3是人卵巢、胃肠道、肺癌和乳腺癌细胞迁移和侵袭所必需的。已经证明MLK3在丝裂原依赖的ERK激活和结肠癌细胞增殖中是必需的；然而，它在结肠癌细胞侵袭中的作用尚不清楚。许多癌细胞具有持续的、内在的氧化应激和比正常细胞更高水平的活性氧物种(ROS)。促炎细胞因子、辐射、毒素和化疗药物等刺激可以触发ROS的产生。当ROS水平很高时，可以激活信号转导通路，诱导细胞凋亡或坏死性死亡；当ROS水平较低时，ROS可以刺激细胞增殖。我们的初步发现表明，氧化应激强烈激活ERK1/2信号，从而促进结肠癌细胞中MLK3的磷酸化。氧化应激还促进依赖ERK的MLK3蛋白水平的增加和MLK3激酶活性的降低。此外，我们还证明了ERK在体外可以使MLK3磷酸化。因此，我们已经确定ERK是MLK3蛋白水平的一种新的正调节因子，以及氧化应激反应中MLK3激酶活性的负调节因子。我们推测，氧化应激反应中失活的MLK3蛋白的增强可能导致持续的MLK3依赖的ERK信号的刺激，从而促进结肠癌细胞的转化和增殖。本研究的总体目标是明确氧化应激诱导ERK依赖的MLK3调控的机制，并阐明这种调控对MLK3依赖的刺激结肠癌细胞转化、增殖和凋亡的影响。因此，该提案的具体目的是：1)研究ERK信号在H202介导的结肠癌细胞MLK3上调中的作用。2)确定ROS和ERK激活对MLK3激酶活性和功能的影响；3)阐明MLK3在结肠癌细胞增殖和侵袭中的作用。这项建议中描述的实验将使我们深入了解氧化应激如何调节MLK3蛋白水平和激酶活性，以及这如何影响MLK3依赖的MAPK信号的激活，以及结肠癌细胞的增殖和侵袭。此外，我们将研究新的策略，以耗尽细胞MLK3蛋白水平，以抑制结肠癌细胞的增殖和侵袭。

 DESCRIPTION (provided by applicant): Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase (MAP3K) that regulates multiple MAPK signaling pathways in response to mitogenic and stress stimuli. Depending on the cellular context, MLK3 can phosphorylate MKK4 or MKK7 MAPK kinases (MAP2Ks), which activate c-Jun N-terminal Kinase (JNK) to trigger cell death. Alternatively, MLK3, through a kinase-independent scaffold function, can activate B-Raf and extracellular signal-regulated kinase (ERK) signaling to promote cell proliferation. MLK3 is required for the migration and invasion of human ovarian, gastrointestinal, lung, and breast cancer cells. A requirement for MLK3 in mitogen-dependent ERK activation and colon cancer cell proliferation has been demonstrated; however its function in colon cancer cell invasion is not known. Many cancer cells have persistent, intrinsic oxidative stress and higher levels of reactive oxygen species (ROS) than normal cells. Stimuli such as pro-inflammatory cytokines, irradiation, toxins and chemotherapeutic drugs can trigger ROS production. At very high levels ROS can elicit activation of signal transduction pathways and induce apoptosis or necrotic cell death, and at low levels ROS can stimulate cell proliferation. Our preliminary findings indicate that oxidative stress strongly activates ERK1/2 signaling which promotes the phosphorylation of MLK3 in colon cancer cells. Oxidative stress also promotes an ERK-dependent increase in MLK3 protein level and a decrease in MLK3 kinase activity. Furthermore, we demonstrated that ERK could phosphorylate MLK3 in vitro. Thus, we have identified ERK as a novel, positive regulator of MLK3 protein level and a negative regulator of MLK3 kinase activity in response to oxidative stress. We postulate that the enhancement of inactive MLK3 protein in response to oxidative stress could cause persistent MLK3-dependent stimulation of ERK signaling and thereby promote cellular transformation and proliferation of colon cancer cells. The overall goal of this proposal is to define the mechanism by which oxidative stress induces ERK-dependent regulation of MLK3, and elucidate the impact of this regulation on MLK3-dependent stimulation of cellular transformation, proliferation and apoptosis in colon cancer cells. Accordingly the specific aims of the proposal are: 1) To investigate the role of ERK signaling in H202-mediated upregulation of MLK3 in colon cancer cells. 2) To determine the impact of ROS and ERK activation on MLK3 kinase activity and function, 3) To elucidate the function of MLK3 in colon cancer cell proliferation and invasion. The experiments described in this proposal will allow us to gain a thorough understanding of how oxidative stress modulates MLK3 protein level and kinase activity and how this affects MLK3-dependent activation of MAPK signaling, proliferation and invasion of colon cancer cells. Furthermore, we will examine novel strategies to deplete cellular MLK3 protein levels to inhibit colon cancer cell proliferation and invasion.