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

描述（由申请人提供）：NF 2患者表现出NF 2肿瘤抑制基因的功能缺失突变或缺失。NF 2基因编码一种595个氨基酸的肿瘤抑制蛋白，称为Merlin。Merlin与Ezrin、Radixin和Moesin（ERM）组细胞蛋白具有同源性，所述细胞蛋白将整合膜蛋白连接至肌动蛋白细胞骨架。Rac和Cdc 42是Rho蛋白家族的小GTP酶，是细胞骨架的关键调节剂，Merlin被认为是Rac依赖性信号传导的抑制剂。Merlin还抑制细胞生长。Merlin的结合配偶体在其生长抑制功能中可能至关重要。我们已经确定了梅林和混合谱系激酶3（MLK 3）之间的一种新的相互作用。MLK 3是一种丝氨酸/苏氨酸激酶，可激活多种MAP激酶信号通路。Cdc 42和Rac是MLK 3的上游激活剂。我们的研究表明Merlin是MLK 3的生理抑制剂。我们推测Merlin通过抑制MLK 3活化MAPK信号传导来阻断Rac依赖性信号传导。此外，NF 2肿瘤细胞中功能性Merlin的缺乏可能导致活性MLK 3的基础水平增加，这可能促进细胞生长和细胞转化。本提案的目的是分析Merlin和MLK 3之间相互作用的功能意义，研究NF 2表达的丧失是否增强MLK 3、ERK和JNK激酶活性，并确定Merlin是否抑制MLK 3转化细胞和人肿瘤细胞的细胞增殖和恶性转化特征。我们将对全长和截短的、过表达的MLK 3和Merlin蛋白进行免疫共沉淀，以鉴定Merlin和MLK 3相互作用所需的特异性区域。将采用间接免疫荧光研究来研究MLK 3和Merlin在雪旺细胞中的共定位。将在存在或不存在共表达Merlin的情况下分析MLK 3与其上游激活剂的结合。将使用正常细胞、缺乏NF 2表达的肿瘤细胞、NF 2表达受调节的细胞和其中NF 2表达已被沉默的细胞来分析Merlin对MLK 3、ERK和JNK活化的影响。将在MLK 3转化的NIH-3 T3细胞、SKOV 3卵巢肿瘤细胞和HEI-193肿瘤细胞中研究Merlin对MLK 3介导的细胞转化的影响。将用过表达Merlin或缺乏MLK 3结合区的Merlin的慢病毒感染细胞，并分析细胞增殖、锚定非依赖性生长和细胞侵袭性。总的来说，本研究的结果将使我们能够定义Merlin-MLK 3相互作用的生化意义以及Merlin对MLK 3依赖性信号传导和细胞转化的影响。我们已经确定了一种新的相互作用之间的梅林，蛋白质产物的NF 2肿瘤抑制基因，MLK 3，一个MAP 3 K，调节多个MAPK途径。该提案的重点是破译Merlin在调节MLK 3依赖性MAPK信号传导、细胞增殖和细胞转化中的功能。由于沉默MLK 3抑制NF 2人神经鞘瘤和NF 2-/-小鼠骨肉瘤细胞的增殖，MLK 3可能是开发治疗NF 2肿瘤的疗法的有希望的新靶点。总的来说，这项研究的结果将是我们目前对Merlin功能的理解的重要贡献，将使我们对NF 2中的MAPK信号传导有价值的见解，并将扩大我们对潜在信号传导蛋白的了解，这些蛋白可以用于开发NF 2患者的治疗方法。

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.