Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2.
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.
Patrakitkomjorn
Siriporn
S
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University School of Medicine, Kumamoto, Japan.
Kobayashi
Daiki
D
Morikawa
Takashi
T
Wilson
Masayo Morifuji
MM
Tsubota
Nobuyuki
N
Irie
Atsushi
A
Ozawa
Tatsuya
T
Aoki
Masashi
M
Arimura
Nariko
N
Kaibuchi
Kozo
K
Saya
Hideyuki
H
Araki
Norie
N
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020932:Nerve Growth Factor / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000473:pathology; D009456:Neurofibromatosis 1 / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D025542:Neurofibromin 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D040901:Proteomics / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D015854:Up-Regulation / Q000187:drug effects; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism
D020690:GTPase-Activating Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D025542:Neurofibromin 1; D034741:RNA, Small Interfering; C095114:collapsin response mediator protein-2; D020932:Nerve Growth Factor; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D054460:rho-Associated Kinases; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; C495907:Cdk5 protein, rat; D038362:Glycogen Synthase Kinase 3; D015689:Oncogene Protein p21(ras)