Role of Mixed Lineage Kinase 3/SPRK in Stress Signaling

描述（由申请人提供）：MLK3，也被称为SPRK，是一种丝裂原激活的蛋白激酶，可以激活c-jun N-末端激酶（JNK）并诱导神经元和非神经元细胞的细胞死亡。MLK家族成员的广谱抑制剂（CEP-1347）正在临床试验中，用于治疗神经退行性疾病相关疾病，如帕金森病。TNF-a和神经酰胺已被证明在神经退行性疾病中引起细胞死亡。有趣的是，神经酰胺也已成为化学和放射治疗反应中细胞死亡的生化介质。我们实验室的研究表明，在哺乳动物细胞中，细胞死亡和分化诱导配体、TNF-a和神经酰胺是MLK3的有效激动剂。此外，我们最近的研究（包括已发表的和未发表的）表明(i)神经酰胺可以直接关联并激活MLK3， （ii） MLK3介导TNF-a和神经酰胺诱导的JNK激活，（iii） TNF-a处理Jurkat细胞触发TRAF2与MLK3的关联，（vi） MLK3过表达导致细胞死亡，(v)视黄酸诱导的HL-60细胞分化被mlk特异性抑制剂抑制。基于这些观察结果，我们的主要目标是阐明神经酰胺和TNF-a激活MLK3的机制，并确定配体激活的MLK3在细胞死亡和分化途径中的生理作用。这些目标将实现以下四个具体目标。在目的1中，将阐明神经酰胺激活MLK3的体内外机制。在目标2中，将研究tnf -a诱导的MLK3激活的机制，以及它与神经酰胺诱导的MLK3激活的协同作用。在目标3中，MLK3在介导细胞死亡和分化途径中的生理作用将通过生化和遗传方法来阐明。在目的4中，我们将利用新近创建的MLK3 KO和野生型动物，在MPTP小鼠模型（即PD模型）中研究MLK3在神经元凋亡中的生理作用。阐明神经酰胺和tnf诱导的MLK3激活的机制，以及MLK3在调节细胞死亡和分化途径中的作用，将为开发针对神经退行性、创伤或缺血等疾病导致的病理性神经元损失的靶向治疗干预提供基础。此外，这些研究的结果将有助于开发新的药理学方法，以提高癌症治疗的化疗或放射增敏的效率和特异性。

DESCRIPTION (provided by applicant): MLK3, also known as SPRK, is a mitogen-activated protein kinase that can activate c-jun N- terminal kinase (JNK) and induce cell death in neuronal and non-neuronal cells. A broad-spectrum inhibitor of MLK family members (CEP-1347) is under clinical trials for treating neurodegenerative disorder related diseases like Parkinson's. TNF-a and ceramide have been shown to cause cell death during neurodegenerative disorders. Interestingly, ceramide has also emerged as a biochemical mediator of cell death in response to chemo- and radio- therapies. Studies from our laboratory have revealed that the cell death and differentiation inducing ligands, TNF-a and ceramide are potent agonists of MLK3 in mammalian cells. In addition, our more recent studies (both published and unpublished) indicate that (i) ceramides can directly associate, and activate MLK3, (ii) MLK3 mediates TNF-a and ceramide-induced JNK activation, (iii) TNF-a treatment of Jurkat cells triggers TRAF2 association with MLK3, (vi) overexpression of MLK3 causes cell death, and (v) Retinoic acid induced HL-60 cell differentiation is inhibited by MLKs specific inhibitor. Based on these observations, our major goals for this grant application are to elucidate the mechanisms of MLK3 activation by ceramide and TNF-a, and to define the physiological roles of ligand-activated MLK3 in cell death and differentiation pathways. These goals will be achieved following four specific aims. In aim 1, the in vitro and in vivo mechanisms of MLK3 activation by ceramide will be elucidated. In aim 2, the mechanism of TNF-a-induced MLK3 activation, and its synergy with ceramide-induced MLK3 activation will be examined. In aim 3, the physiological roles of MLK3 in mediating cell death and differentiation pathways will be elucidated using both biochemical and genetic approaches. In aim 4, the physiological role of MLK3 in neuronal apoptosis will be examined in MPTP mouse model (i.e. PD model), using recently created MLK3 KO and wild type animals. Elucidating the mechanism(s) of ceramide and TNF-ainduced MLK3 activation, and MLK3's role in modulating cell death and differentiation pathways will offer a basis for the development of targeted therapeutic interventions for pathologic neuronal loss occurring as a result of conditions, such as neurodegeneration, trauma or ischemia. Furthermore, the results from these studies will aide in the development of novel pharmacological approaches towards increasing the efficiency and specificity of chemo- or radio- sensitization for cancer therapies.