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.