| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2005: ¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 2004: ¥7,100,000 (Direct Cost: ¥7,100,000)
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| Research Abstract |
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
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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
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