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MLK4 regulation of MAPK signaling

MLK4 对 MAPK 信号传导的调节

基本信息

批准号：
8366331
负责人：
Deborah N Chadee
金额：
$ 34.92万
依托单位：
UNIVERSITY OF TOLEDO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2016-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Mixed Lineage Kinases (MLKs) are a family of mitogen-activated protein (MAP) kinase kinase kinases (MAP3Ks) that activate multiple MAP kinase (MAPK) signaling pathways to elicit specific cellular responses. The MLK subfamily consists of MLKs 1, 2, 3, 4 and 4. Of these, MLK3 has been the best characterized in terms of biological function and biochemical activity. MLK3 activates ERK, JNK and p38 MAPK pathways and has important functions in cell proliferation and tumor cell invasion. In contrast, very little is known about the biological functon and biochemical activity of MLK4, for which there are two alternatively spliced isoforms. Our preliminary results suggest that MLK4 does not function as a typical MAPK in promoting activation of MAPK signaling pathways. Instead MLK4 is a negative regulator of MAPK signaling and tumor cell invasion. Furthermore, we have identified a novel interaction between MLK4 and MLK3, and we propose that MLK4 suppresses MAPK signaling and invasion by directly inhibiting MLK3 activation. The experiments described in this proposal are designed to investigate the function of MLK4 in regulating MLK3 activation, MAPK signaling and tumor cell invasion. Accordingly, the specific aims of this proposal are as follows: (1) To analyze the role o MLK4 in regulating MAPK signaling. In vitro kinase assays will be performed to test if MLK4 has activity towards MAP2Ks, and if MLK4 inhibits the activity of other MAP3Ks in addition to MLK3. (2) To investigate the MLK3-MLK4 interaction and the mechanism by which MLK4 inhibits MLK3 activation. In vitro binding assays will be performed with MLK4 and MLK3 deletion mutants to identify the regions of MLK3 and MLK4 that interact. Co-immunoprecipitations will be performed in cells overexpressing MLK4 to determine if MLK4 inhibits the MLK3-TRAF, MLK3-Cdc42 or MLK3-B-Raf interactions. (3) To investigate the function of MLK4 in tumor cell proliferation and invasion. Cell proliferation assays and invasion assays (using a modified Boyden chamber) will be performed to test if MLK4 expression or knockdown affects cell proliferation or invasion. The results from the proposed studies will broaden our understanding of the different roles of MLKs in cell signaling and tumor cell invasion, which could provide insight into how altered MLK activity may contribute to establishment of the malignant phenotype. PUBLIC HEALTH RELEVANCE: MAPK signaling pathways have a central role in the regulation of numerous cellular responses such as proliferation, differentiation, migration and invasion. Since persistent activation of MAPK signaling is a major contributor to the establishment of a malignant phenotype, a thorough understanding of how MAPK pathways are tightly controlled by both positive and negative regulatory factors is essential to the development of novel cancer therapies.

描述（由申请人提供）：混合谱系激酶（MLK）是促分裂原活化蛋白（MAP）激酶（MAP 3 K）家族，其激活多个MAP激酶（MAPK）信号通路以引发特异性细胞应答。MLK亚家族由MLK 1、2、3、4和4组成。其中，MLK 3在生物学功能和生物化学活性方面已被最好地表征。MLK 3激活ERK、JNK和p38 MAPK通路，并在细胞增殖和肿瘤细胞侵袭中具有重要功能。相反，MLK 4的生物学功能和生化活性知之甚少，MLK 4有两种选择性剪接异构体。我们的初步结果表明，MLK 4不作为一个典型的MAPK在促进MAPK信号通路的激活。相反，MLK 4是MAPK信号传导和肿瘤细胞侵袭的负调节剂。此外，我们已经确定了MLK 4和MLK 3之间的一种新的相互作用，我们提出MLK 4通过直接抑制MLK 3活化来抑制MAPK信号传导和侵袭。本实验旨在研究MLK 4在调节MLK 3活化、MAPK信号传导和肿瘤细胞侵袭中的作用。因此，本研究的具体目的如下：（1）分析MLK 4在MAPK信号转导中的作用。将进行体外激酶测定以测试MLK 4是否对MAP 2K具有活性，以及MLK 4是否抑制除MLK 3之外的其他MAP 3 K的活性。(2)研究MLK 3-MLK 4相互作用以及MLK 4抑制MLK 3活化的机制。将使用MLK 4和MLK 3缺失突变体进行体外结合试验，以鉴定MLK 3和MLK 4相互作用的区域。将在过表达MLK 4的细胞中进行免疫共沉淀，以确定MLK 4是否抑制MLK 3-TRAF、MLK 3-Cdc 42或MLK 3-B-Raf相互作用。(3)探讨MLK 4在肿瘤细胞增殖和侵袭中的作用。将进行细胞增殖试验和侵袭试验（使用改良的Boyden室），以检测MLK 4表达或敲低是否影响细胞增殖或侵袭。拟议研究的结果将拓宽我们对MLK在细胞信号传导和肿瘤细胞侵袭中的不同作用的理解，这可以深入了解MLK活性的改变如何有助于恶性表型的建立。公共卫生关系： MAPK信号通路在许多细胞反应如增殖、分化、迁移和侵袭的调节中具有核心作用。由于MAPK的持续激活，由于MAPK信号是恶性表型形成的主要因素，因此深入了解MAPK通路如何受到正性和负性调节因子的严格控制对于开发新型癌症疗法至关重要。