Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2

J Biol Chem. 2008 Apr 4;283(14):9399-413. doi: 10.1074/jbc.M708206200. Epub 2008 Jan 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / drug effects
  • Cell Differentiation / genetics
  • Cyclin-Dependent Kinase 5 / genetics
  • Cyclin-Dependent Kinase 5 / metabolism
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism
  • Glycogen Synthase Kinase 3 / genetics
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • Nerve Growth Factor / pharmacology
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurites / metabolism*
  • Neurites / pathology
  • Neurofibromatosis 1 / genetics
  • Neurofibromatosis 1 / metabolism
  • Neurofibromatosis 1 / pathology
  • Neurofibromin 1 / antagonists & inhibitors
  • Neurofibromin 1 / genetics
  • Neurofibromin 1 / metabolism*
  • Oncogene Protein p21(ras) / genetics
  • Oncogene Protein p21(ras) / metabolism
  • PC12 Cells
  • Phosphorylation / drug effects
  • Protein Structure, Tertiary / genetics
  • Proteomics / methods
  • RNA, Small Interfering / genetics
  • Rats
  • Up-Regulation / drug effects
  • rho-Associated Kinases / genetics
  • rho-Associated Kinases / metabolism

Substances

  • GTPase-Activating Proteins
  • Intercellular Signaling Peptides and Proteins
  • Nerve Tissue Proteins
  • Neurofibromin 1
  • RNA, Small Interfering
  • collapsin response mediator protein-2
  • Nerve Growth Factor
  • Cyclin-Dependent Kinase 5
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, mouse
  • Gsk3b protein, rat
  • rho-Associated Kinases
  • CDK5 protein, human
  • Cdk5 protein, mouse
  • Cdk5 protein, rat
  • Glycogen Synthase Kinase 3
  • Oncogene Protein p21(ras)