Neurofibromatosis Type 1 (NF1) Tumor Suppressor, Neurofibromin, Regulates the Neuronal Differentiation of PC12 Cells via Its Associating Protein, CRMP-2

Masayo Morifuji Wilson,Masashi Aoki,Takashi Morikawa,Atsushi Irie,Norie Araki,Daiki Kobayashi,Nobuyuki Tsubota,Kozo Kaibuchi,Siriporn Patrakitkomjorn,Hideyuki Saya,Tatsuya Ozawa,Nariko Arimura

doi:10.1074/jbc.m708206200

Masayo Morifuji Wilson, Masashi Aoki + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m708206200

Copy DOI

Abstract

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 CRMP-2 (Collapsin response mediator protein-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-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, 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.

Highlights

The Neurofibromatosis type 1 (NF1) gene locates on chromosome 17q11.2 and encodes a large protein of 2,818 amino acids, neurofibromin [2]
Because the great majority of NF1 gene mutations frequently found in NF1 patients prevents 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]
We demonstrated a novel role for neurofibromin on neuronal differentiation in conjunction with regulation of Ras activity via its GAP-related domain (GRD) in NGF-stimulated PC12 cells serving as a model for neuronal cells [6]

Summary

EXPERIMENTAL PROCEDURES

Preparation of Glutathione S-transferase (GST) Fusion Proteins, Plasmid Constructions, and Transfections—GST fusion domain proteins of human neurofibromin corresponding to sequences of residues 543–909, 1168 –1530, and 2260 –2818 of neurofibromin, which were designated cysteine/serine-rich domain (CSRD), GAP-related domain (GRD), and C-terminal domain (CTD), respectively, were produced in Escherichia coli under the isopropyl 1-thio-␤-D-galactopyranoside induction system and affinity-purified as described previously [10]. After reaction with horseradish peroxidaseor Cy5-conjugated secondary antibodies, the reacted protein pattern on the membrane was visualized by an ECL detection system or by scanning with fluorescent scanner Typhoon 9400 (GE Healthcare), respectively. CRMP-2 protein (500 ␮g) was prepared after the thrombin (48 units) treatment of GST-CRMP-2 in thrombin cleavage buffer (50 mM Tris-HCl, pH 8.8, 150 mM NaCl, 2.5 mM CaCl2) for 40 min at room temperature, followed by the gel filtration. Two-dimensional Electrophoresis and Two-dimensional Difference Gel Electrophoresis (DIGE)—Mouse brain or PC12 cell lysates (10 –50 ␮g), after desalting using two-dimensionalclean up kit (Amersham Biosciences), were mixed with 125 ␮l of rehydration solution (8 M urea, 0.5% (w/v) CHAPS, 0.2% (w/v) DTT, 0.5% (v/v) IPG buffer) and loaded into strip holders for first-dimension isoelectric focusing. The fluorescent images were scanned with Typhoon 9400, visualized, and processed as digital data with data mining software ImageQuant version 5.2, DeCyder (GE Healthcare), and ProGenesis Work station version 2005 (PerkinElmer Life Sciences)

RESULTS

60 S ribosomal protein L22

DISCUSSION