Role of Mixed Lineage Kinase 3/SPRK in Stress Signaling

描述(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氨基末端激酶(JNK)，并诱导神经元和非神经元细胞的细胞死亡。一种广谱的MLK家族成员抑制剂(CEP-1347)正在进行临床试验，用于治疗与神经退行性疾病相关的疾病，如帕金森氏症。肿瘤坏死因子-α和神经酰胺已被证明在神经退行性疾病中导致细胞死亡。有趣的是，神经酰胺也已经成为细胞死亡的生化媒介，对化疗和放射治疗的反应。我们实验室的研究表明，细胞死亡和分化诱导配体、肿瘤坏死因子-α和神经酰胺是哺乳动物细胞中MLK3的强有力的激动剂。此外，我们最近发表和未发表的研究表明：(I)神经酰胺可以直接结合并激活MLK3，(Ii)MLK3介导人肿瘤坏死因子-α和神经酰胺诱导的JNK活化，(Iii)肿瘤坏死因子-α处理Jurkat细胞触发TRAF2与MLK3的关联，(Vi)过表达MLK3导致细胞死亡，(V)维甲酸诱导的HL-60细胞分化被MLKs特异性抑制剂抑制。基于这些观察，我们这次拨款申请的主要目标是阐明神经酰胺和肿瘤坏死因子-α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。这些目标将在以下四个具体目标下实现。目的1阐明神经酰胺激活MLK3的体内外机制。在目标2中，将研究肿瘤坏死因子-α诱导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，将用生化和遗传学方法阐明MLK3在介导细胞死亡和分化途径中的生理作用。在目的4中，将利用新近建立的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。阐明神经酰胺和肿瘤坏死因子诱导的MLK3激活的机制(S)，以及MLK3的S在调节细胞死亡和分化途径中的作用，将为开发针对因神经变性、创伤或缺血等原因而导致的病理性神经元丢失的靶向治疗干预措施提供基础。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 In addition, our more recent studies (both published and unpublished) indicate that (i) ceramides can directly associate, and activate MLK3, (ii) MLK3 mediates TNF-a and ceramide-induced JNK activation, (iii) TNF-a treatment of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.