Study for the cellular signal transduction of Neurofibromatosis Type 1 and Type 2 tumor suppressor gene products to establish the medical molecular target for NF1 and NF2

神经纤维瘤病1型（NF1）和2型（NF2）是常染色体显性遗传疾病，与颅内肿瘤的发展密切相关，包括双侧前庭神经鞘瘤、脑膜瘤和胶质瘤。为了阐明NF1和NF2基因产物的生物学功能，我们利用siRNA技术和蛋白质组学策略分析了它们的细胞信号转导。1) NF1;NF1 siRNA诱导特征性形态变化，如过度的肌动蛋白应激纤维形成，伴随着coffin磷酸化水平的升高，通过解聚和切断肌动蛋白丝来调节肌动蛋白细胞骨架重组，并通过激活Rho-ROCK-LIMK2途径促进这一过程，这需要Ras激活。采用定量差异蛋白质组学策略（iTRAQ）鉴定了53种与神经纤维蛋白相关的蛋白。这些蛋白包括坍缩反应介质蛋白-2 （CRMP-2）、钙调蛋白（CaM）、14-3-3蛋白、Micr…More小管相关蛋白、coffilin等。在PC12细胞中，CRMP2和微管蛋白与神经纤维蛋白的关联受其磷酸化方式调节。NF1 SiRNA显著抑制PC12细胞的神经突延伸，这与GSK-3b、rho激酶等激酶调控CRMP-2磷酸化有关。神经纤维蛋白在调节细胞骨架重组机制中具有重要作用，影响细胞运动、粘附和分化。2) NF2;鉴定出了28个梅林的细胞结合蛋白，重点是DNA修复酶，如聚adp核糖聚合酶、DNA- pk亚基ku -抗原85和70。merlin的n端（19-339）区域对它们的相互作用至关重要。merlin和PARP的亚细胞共定位主要发生在它们的核区，并依赖于merlin的核输出信号（NES）。这种核积累随着细胞DNA损伤而增加，而MEF （PARP-/-）不能在其核区积累merlin。这些结果表明，merlin是一种细胞质-核穿梭蛋白，其依赖nes的转运途径与PARP相关。NF蛋白通过其细胞相关蛋白进行功能调控是必不可少的，NF基因畸变引起的NF蛋白异常调控可能与NF相关的发病机制有关。少

The neurofibromatosis type 1 (NF1) and type 2 (NF2) are autosomal dominantly inherited disorders, and strongly associated with development of intracranial tumors including bilateral vestibular schwannomas, meningiomas and gliomas. To elucidate the biological function of NF1 and NF2 gene products, and, we analyzed their cellular signal transductions by using siRNA techniques and proteomic strategies. 1)NF1 ; NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated phosphorylation levels of coffin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments, and that was promoted by the activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation. Fifty three proteins associating to neurofibromin were identified with the quantitative differential proteomic strategy (iTRAQ). These proteins contain collapsin response mediator protein-2 (CRMP-2), calmodulin (CaM), 14-3-3 proteins, Micr … More otubule-associated proteins, coffilin etc. In PC12 cells, the associations of CRMP2 and tubulin to neurofibromin were regulated by their phosphorylation manners. NF1 SiRNA significantly inhibited the neurite extension of PC12 cells, which was correlated with the regulation of CRMP-2 phosphorylations via GSK-3b, Rho-kinase, and other kinases. Neurofibromin has significant roles in the machinery regulating cellular cytoskeletal reorganization, which affects cell motility, adhesion, and differentiations. 2)NF2 ; 28 cellular binding proteins of merlin were identified, and focused on DNA repair enzymes such as poly ADP-ribose polymerase, DNA-PK subunits Ku-antigen 85 and 70. The N-terminal (19-339) region of merlin is essential for their interaction. Subcellular co-localizations of merlin and PARP were observed mainly in their nuclear region with a nuclear export signal (NES) dependent manner of merlin. This nuclear accumulation increased with cellular DNA damages, whereas MEF (PARP-/-) could not accumulate merlin in their nuclear region. These results suggest that merlin is a cytoplasmic-nuclear shuttling protein directed by its NES-dependent transport pathway being associated with PARP. Functional regulations of NF proteins via their cellular associating proteins are essential, and their abnormal regulations caused by NF gene aberrations may be implicated in NF-related pathogenesis. Less