Regulation of Mixed Lineage Kinase 3 by the tumor suppressor protein Merlin

肿瘤抑制蛋白 Merlin 对混合谱系激酶 3 的调节

• 批准号: 7364459
• 负责人: Deborah N Chadee
• 金额: $ 21.6万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7364459
• 关键词: Actins; Affect; Amino Acids; Anchorage-Independent Growth; Binding; Biochemical; Biological; Biological Process; Cell Proliferation; Cells; Characteristics; Co-Immunoprecipitations; Cyclin D1; Cytoskeletal Modeling; Cytoskeleton; Development; Exhibits; Growth; Human; Indirect Immunofluorescence; Integral Membrane Protein; JNK-activating protein kinase; Knowledge; Length; MAP Kinase Gene; MAP Kinase Signaling Pathways; MAPK8 gene; Malignant - descriptor; Mediating; Mitogen-Activated Protein Kinases; Mitogens; Monomeric GTP-Binding Proteins; Mus; NF2 gene; NIH 3T3 Cells; Neurilemmoma; Neurofibromin 2; Normal Cell; Pathway interactions; Patients; Phosphotransferases; Physiological; Play; Property; Protein Family; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Rattus; Regulation; Role; Schwann Cells; Signal Transduction; Signaling Protein; Subfamily lentivirinae; Tetracycline; Tetracyclines; Tumor Suppressor Genes; Tumor Suppressor Proteins; cell growth; cell transformation; ezrin; functional loss; inhibitor/antagonist; insight; link protein; loss of function mutation; metaplastic cell transformation; mixed lineage kinase 3; moesin; neoplastic cell; novel; osteosarcoma; ovarian neoplasm; prevent; radixin protein; research study; rho; rho GTP-Binding Proteins; therapy development; tumor

DESCRIPTION (provided by applicant): Patients with NF2 exhibit loss of function mutations or deletions the NF2 tumor suppressor gene. The NF2 gene encodes a 595 amino acid tumor suppressor protein called Merlin. Merlin has homology to the Ezrin, Radixin and Moesin (ERM) group of cellular proteins that link integral membrane proteins to the actin cytoskeleton. Rac and Cdc42, small GTPases of the Rho family of proteins, are critical regulators of the cytoskeleton and Merlin has been implicated as an inhibitor of Rac-dependent signaling. Merlin also inhibits cell growth. The binding partners for Merlin may be critically important in its growth suppressive function. We have identified a novel interaction between Merlin and Mixed Lineage Kinase 3 (MLK3). MLK3 is a serine/threonine kinase that activates multiple MAP kinase signaling pathways. Cdc42 and Rac are upstream activators of MLK3. Our studies suggest that Merlin is a physiological inhibitor of MLK3. We postulate that Merlin blocks Rac- dependent signaling by inhibiting MLK3 activation of MAPK signaling. Furthermore, the lack of functional Merlin in NF2 tumor cells could cause an increase in the basal level of active MLK3 which may promote cell growth and cellular transformation. The aims of this proposal are to analyze the functional significance of the interaction between Merlin and MLK3, to investigate if loss of NF2 expression augments MLK3, ERK and JNK kinase activities, and to determine if Merlin inhibits cell proliferation and characteristics of malignant transformation of MLK3-transformed cells and human tumor cells. We will perform co- immunoprecipitations of full length and truncated, overexpressed MLK3 and Merlin proteins to identify the specific regions of Merlin and MLK3 that are required for their interaction. Indirect immunofluorescence studies will be employed to study the colocalization of MLK3 and Merlin in schwann cells. Binding of MLK3 to its upstream activators will be analyzed in the presence or absence of co-expressed Merlin. Normal cells, tumor cells lacking NF2 expression, cells that have regulated expression of NF2, and cells in which NF2 expression has been silenced, will be used to analyze the effect of Merlin on MLK3, ERK and JNK activation. The impact of Merlin on MLK3- mediated cellular transformation will be investigated in MLK3-transformed NIH-3T3 cells, SKOV3 ovarian tumor cells and HEI-193 tumor cells. Cells will be infected with a lentivirus that overexpresses Merlin, or Merlin lacking the MLK3 binding region, and cell proliferation, anchorage independent growth, and invasiveness of the cells will be analyzed. Collectively, the results of this study will allow us to define the biochemical significance of the Merlin-MLK3 interaction and the impact of Merlin on MLK3-dependent signaling and cellular transformation. We have identified a novel interaction between Merlin, the protein product of the NF2 tumor suppressor gene, and MLK3, a MAP3K that regulates multiple MAPK pathways. This proposal is focused on deciphering the function of Merlin in regulation of MLK3- dependent MAPK signaling, cell proliferation and cellular transformation. Since silencing mlk3 inhibits proliferation of NF2 human schwannoma and NF2 -/- mouse osteosarcoma cells, MLK3 could be a promising new target for the development of therapies to treat NF2 tumors. Collectively, the results of this study will be an important contribution to our current understanding of Merlin function, will give us valuable insight into MAPK signaling in NF2, and will expand our knowledge of potential signaling proteins that can be targeted for the development of treatments for patients with NF2.

描述（由申请人提供）：NF2患者表现出NF2肿瘤抑制基因突变或缺失的功能丧失。NF2基因编码一种名为Merlin的595个氨基酸的肿瘤抑制蛋白。Merlin与Ezrin， Radixin和Moesin （ERM）组细胞蛋白同源，这些细胞蛋白将整体膜蛋白连接到肌动蛋白细胞骨架上。Rac和Cdc42是Rho蛋白家族的小gtpase，是细胞骨架的关键调节因子，而Merlin被认为是Rac依赖性信号传导的抑制剂。梅林还能抑制细胞生长。Merlin的结合伙伴可能对其生长抑制功能至关重要。我们已经确定了Merlin和混合谱系激酶3 （MLK3）之间的一种新的相互作用。MLK3是一种丝氨酸/苏氨酸激酶，可激活多种MAP激酶信号通路。Cdc42和Rac是MLK3的上游活化剂。我们的研究表明，Merlin是MLK3的一种生理性抑制剂。我们假设Merlin通过抑制MLK3激活MAPK信号传导来阻断Rac依赖的信号传导。此外，NF2肿瘤细胞中缺乏功能性Merlin可能导致活性MLK3基础水平升高，从而促进细胞生长和细胞转化。本课题旨在分析Merlin与MLK3相互作用的功能意义，探讨NF2表达缺失是否会增强MLK3、ERK和JNK激酶活性，确定Merlin是否会抑制细胞增殖以及MLK3转化细胞和人肿瘤细胞的恶性转化特征。我们将对全长和截断的、过表达的MLK3和Merlin蛋白进行共免疫沉淀，以确定Merlin和MLK3蛋白相互作用所需的特定区域。间接免疫荧光研究将用于研究MLK3和Merlin在雪旺细胞中的共定位。在存在或不存在共表达的Merlin的情况下，将分析MLK3与其上游激活剂的结合。我们将利用正常细胞、缺乏NF2表达的肿瘤细胞、NF2表达受到调控的细胞以及NF2表达被沉默的细胞来分析Merlin对MLK3、ERK和JNK活化的影响。我们将在MLK3转化的NIH-3T3细胞、SKOV3卵巢肿瘤细胞和HEI-193肿瘤细胞中研究Merlin对MLK3介导的细胞转化的影响。用过表达Merlin或缺乏MLK3结合区Merlin的慢病毒感染细胞，分析细胞增殖、锚定独立生长和细胞侵袭性。总的来说，本研究的结果将使我们能够定义Merlin- mlk3相互作用的生化意义，以及Merlin对mlk3依赖性信号传导和细胞转化的影响。我们已经确定了NF2肿瘤抑制基因的蛋白产物Merlin和MLK3之间的一种新的相互作用，MLK3是一种调节多种MAPK通路的MAP3K。这一建议的重点是破译梅林在调节MLK3依赖的MAPK信号，细胞增殖和细胞转化中的功能。由于沉默mlk3可以抑制NF2人神经鞘瘤和NF2 -/-小鼠骨肉瘤细胞的增殖，因此mlk3可能是开发治疗NF2肿瘤的有希望的新靶点。总的来说，这项研究的结果将对我们目前对Merlin功能的理解做出重要贡献，将使我们对NF2中的MAPK信号传导有价值的见解，并将扩大我们对潜在信号蛋白的认识，这些信号蛋白可以用于开发NF2患者的治疗方法。

项目成果

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6.

• DOI: 10.1016/j.cellsig.2009.06.008
• 发表时间: 2009-11
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Korchnak AC;Zhan Y;Aguilar MT;Chadee DN
• 通讯作者: Chadee DN

MLK4β functions as a negative regulator of MAPK signaling and cell invasion.

• DOI: 10.1038/oncsis.2012.6
• 发表时间: 2012-03-26
• 期刊: Oncogenesis
• 影响因子: 6.2
• 作者: Abi Saab WF;Brown MS;Chadee DN
• 通讯作者: Chadee DN

Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells.

混合谱系激酶3是基质金属蛋白酶表达和卵巢癌细胞浸润所必需的。

• DOI: 10.1016/j.yexcr.2012.05.002
• 发表时间: 2012-08-15
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: Zhan Y;Abi Saab WF;Modi N;Stewart AM;Liu J;Chadee DN
• 通讯作者: Chadee DN

Regulation of mixed lineage kinase 3 is required for Neurofibromatosis-2-mediated growth suppression in human cancer.

• DOI: 10.1038/onc.2010.453
• 发表时间: 2011-02-17
• 期刊: ONCOGENE
• 影响因子: 8
• 作者: Zhan, Y.;Modi, N.;Stewart, A. M.;Hieronimus, R. I.;Liu, J.;Gutmann, D. H.;Chadee, D. N.
• 通讯作者: Chadee, D. N.