JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration.
Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.
Jin
Junghee
J
Department of Life Science and Medical Bio-Science, Waseda University, Tokyo 162-8480, Japan.
Suzuki
Hiromi
H
Hirai
Syu-Ichi
S
Mikoshiba
Katsuhiko
K
Ohshima
Toshio
T
D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology*; D014162:Transfection
C000714328:DCX protein, human; C000714847:Dcx protein, mouse; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D051360:Cyclin-Dependent Kinase 5; D048031:JNK Mitogen-Activated Protein Kinases