PMID- 10683153 OWN - NLM STAT- MEDLINE DCOM- 20000531 LR - 20220215 IS - 0021-9533 (Print) IS - 0021-9533 (Linking) VI - 113 ( Pt 6) DP - 2000 Mar TI - Brain-derived neurotrophic factor-induced phosphorylation of neurofilament-H subunit in primary cultures of embryo rat cortical neurons. PG - 1059-68 AB - Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in rat embryonic brain neurons in culture. A portion of the NF-H was phosphorylated in vivo at embryonic day 17 when brain neurons were prepared. When the neurons were isolated and cultured, the NF proteins disappeared once and then reappeared over the next several days in the following order: (1) NF-L/NF-M, (2) dephosphorylated NF-H and (3) phosphorylated NF-H. Phosphorylation of NF-H began around 4 days after cell plating, at about the time of synapse formation. Treatments that appeared to modulate the timing of synapse formation also affected the timing of NF-H phosphorylation: (1) earlier phosphorylation was observed at higher neuronal cell density, (2) earlier phosphorylation was observed in neurons cultured on a coating substrate that promotes rapid neurite extension and (3) phosphorylation was suppressed when neurite extension was inhibited by brefeldin A. Three possible synapse formation-induced events, excitation, cell-cell contact through adhesion proteins and elevated concentrations of neurotrophic factors, were examined for their possible involvement in generating the signal for NF-H phosphorylation. Neither excitation nor cell contact enhanced NF-H phosphorylation. Neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulated phosphorylation of NF-H. The BDNF-stimulated phosphorylation was inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase. Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress-activated protein kinase (SAPK), CDK5 and SAPK showed an increase in kinase activity or an active form with a time course similar to NF-H phosphorylation in control culture. On the other hand, BDNF stimulated the kinase activity of CDK5 and induced appearance of an active form of ERK transiently. These results suggest a possibility that synapse formation induces NF-H phosphorylation, at least in part, through activation of CDK5 by BDNF. FAU - Tokuoka, H AU - Tokuoka H AD - Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. hisanaga-shinichi@c.metro-u.ac.jp FAU - Saito, T AU - Saito T FAU - Yorifuji, H AU - Yorifuji H FAU - Wei, F AU - Wei F FAU - Kishimoto, T AU - Kishimoto T FAU - Hisanaga, S AU - Hisanaga S LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - J Cell Sci JT - Journal of cell science JID - 0052457 RN - 0 (Brain-Derived Neurotrophic Factor) RN - 0 (Neurofilament Proteins) RN - 108688-71-7 (neurofilament protein H) SB - IM MH - Animals MH - Brain-Derived Neurotrophic Factor/*metabolism MH - Cell Adhesion MH - Cells, Cultured MH - Cerebral Cortex/*metabolism MH - Neurofilament Proteins/*metabolism MH - Neurons/*metabolism MH - Phosphorylation MH - Rats MH - Signal Transduction MH - Synapses EDAT- 2000/02/22 09:00 MHDA- 2000/06/03 09:00 CRDT- 2000/02/22 09:00 PHST- 2000/02/22 09:00 [pubmed] PHST- 2000/06/03 09:00 [medline] PHST- 2000/02/22 09:00 [entrez] AID - 10.1242/jcs.113.6.1059 [doi] PST - ppublish SO - J Cell Sci. 2000 Mar;113 ( Pt 6):1059-68. doi: 10.1242/jcs.113.6.1059.