1 NF1 TUMOR SUPPRESSOR, NEUROFIBROMIN, REGULATES THE NEURONAL DIFFERENTIATION OF PC12 CELLS VIA ITS ASSOCIATING PROTEIN, COLLAPSIN RESPONSE MEDIATOR PROTEIN-2* Siriporn Patrakitkomjorn 1 , Daiki Kobayashi 1 , Takashi Morikawa 1 , Masayo Morifuji Wilson 1 , Nobuyuki Tsubota 1 , Atsushi Irie 2 , Tatsuya Ozawa 1 , Masashi Aoki 1 , Nariko Arimura 3 , Kozo Kaibuchi 3 , Hideyuki Saya 1 , and Norie Araki 1¶ From 1 The Department of Tumor Genetics and Biology, and 2 The Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan. 3 The Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan Running title: Neurofibromin regulates neuronal differentiation via association with CRMP-2 ¶ Address correspondence to: Norie Araki, Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan Tel: +81-96-373-5323, Fax: +81-96-373-5120, E-mail: nori@gpo.kumamoto-u.ac.jp Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator Collapsin response mediator protein-2 (CRMP-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor (NGF)-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 siRNA significantly inhibited this neurite outgrowth and upregulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b and Rho-kinase (RhoK). Overexpression of the NF1-RAS-GAP related domain rescued these NF1 siRNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. Firstly, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation, and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis. Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disorder, with an estimated prevalence of one in 3-4,000 people (1). The hallmarks of NF1 include development of benign tumors of the peripheral nervous system and an increased risk of developing malignancies. The phenotype of NF1 is highly variable, with several organ systems being affected including the bones, skin, irises and central and peripheral nervous systems. The effects on the nervous system manifest as neurofibroma, gliomas and learning disabilities. The NF1 gene locates on chromosome 17q11.2 and encodes a large protein of 2,818 amino acids, neurofibromin (2). Since the great majority of NF1 gene mutations frequently found in NF1 patients prevent the expression of intact neurofibromin, functional disruption of neurofibromin is potentially relevant to the expression of some or all of the multiple abnormalities that occur in NF1 patients (3). A region centered around 360 amino acid residues encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP). This region is also similar to http://www.jbc.org/cgi/doi/10.1074/jbc.M708206200 The latest version is at JBC Papers in Press. Published on January 23, 2008 as Manuscript M708206200 Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.