BDNF Augments The Phosphorylation Level Of MLC2 Via SRC Signal Pathway In Hippocampal Neurons

BackgroudMyosin family belongs to the motor protein superfamily,which can convert the intracellular energy into chemical energy,and then participates in a series of physiological and biochemical reactions.More than 20 myosin molecules have been found,they have different structures and functions.They have been shown to interact with actin,hydrolyze ATP and produce movement in all cases.Phylogenetic analysis places myosins into 15 classes.Myosin II as the conventional myosins was first found in muscle.The energy produced by hydrolysis of ATP to mediate muscle contraction.With the deepening of research,myosin II distributes in the non-muscle tissues,known as non-muscle Myosin II(non-muscle myosin II,NMII),example neurons.Myosin II is a hexamer and contains a pair of high molecular weight heavy chains(MHC)and two pairs of light molecular weight light chains(MLC)that bind to the heavy chain neck,which contain two regulatory light chains and two essential Light chains.In the central nervous system,Myosin II expresses widely and plays an important role in the migration of neurons,neuritis growth and guidance,microtubule transport in neuronal growth cone,actin dynamic,formation of dendrites spines.Myosin light chain kinase and Rho kinase modulate the activity of myosin light chains.NMII is also involved in the brain's advanced function as the motor proteins.Myosin II plays an important role in the maintenance of Long Term Potentiation(LTP),long-term memory consolidation and the extinction of conditional taste of aversion.All of these reports,they usually infuses shRNA or inhibitors of high weight chains and ATPase activity to silence function of myosin II.But it is not known that how the regulation of myosin ? participate.Brain-derived neurotrophic factor(BDNF)is an important member of neurotrophic factors,it participates in the activities of cell and brain via combining with two kinds of receptors---TrkB and P75NTR to activate downstream signaling pathways.BDNF not only promotes neurons survival and differentiation,dendrites and axonal growth,but also plays important roles in regulating of synaptic plasticity.Synaptic plasticity is usual be identified as the molecular basis of learning and memory,which mediates information transfer between neurons.BDNF could induce LTP formation and maintain in cultural hippocampal neurons.BDNF also can regulate actin dynamic to promote the formation of dendritic spines.Dendritic spines are identified as specially structure of dendrites distributing on dendrites of neurons,which can involve in chemical signal transduction.But it is not known that BDNF whether or not regulate the activity of myosin ? light chain,which pathway involve in this regulation.In this article,we demonstrated that BDNF/TrkB pathway could regulate the activity of Myosin ?,and this up-regulation of phosphorylation myosin ? light chain didn' t depend on three conditional pathways,but SRC family kinase involved.We provided new cognition about the function and mechanism of myosin ?.Methods1.Constructed siRNA2.Specific peptide of MLCK3.Hippocampal neurons cultures and transfection4.Western bolt and CO-ImmunoprecipitationResults1.BDNF regulates MLC2 phosphorylation through high affinity receptor TrkBBy western blot,we demonstrated that gray-scale value of p-MLC2 obviously increased following BDNF stimulation 30 min compared with con group(p<0.05)in cultured hippocampal neurons,and as BDNF stimulation 2 hours,gray-scale value of p-MLC2 continue rising(p?0 01).Cultured hippocampal neurons were no serum starved 10 hours and pretreated with Pep5 or k252a for 30 min,then BDNF stimulates for 2 hours and cells were lysed with TNE buffer.We found that gray value of K252a/BDNF group didn' t increase significantly.Equally,we infused into hippocampus with BDNF,K252a respectively,we found that the protein level of p-MLC2 raised significantly(p<0.05).2.BDNF regulates p-MLC2 phosphorylation through activating MLCKCultured hippocampus neurons were no serum starved 10 hours and pretreated with CalA(inhibitor of phosphatase),Y27623(inhibitor of ROCK kinase),ML-7(inhibitor of MLCK)half an hour and then BDNF was added for 2 hours.The protein of p-MLC2 didn' t changed obviously in ML-7/BDNF group compared with con group.We found that with breaking out the interaction between MLCK and MLC2 by special peptide,BDNF didn' t up-regulate p-MLC2 phosphorylation level.3.BDNF up-regulates p-MLC2 phosphorylation level via SRC pathwayWe found that BDNF regulated p-MLC2 phosphorylation level dependent on activating TrkB receptor,and as known there are three conditional signaling pathway downstream BDNF/TrkB.So we added inhibitors of three conditional signaling U0126,U73122,LY294002 respectively in vitro cultured hippocampus neurons starving 10 hours 30 min before BDNF stimulated for 2 hours.We found that p-MLC2 level didn' t change significantly compared with con group(p>0.05).Next we added PP1(inhibitor of SRC signal)before BDNF stimulating,p-MLC2 protein level reduced significantly compared with con group(p?0.05).We found that knowing down of SRC family members LYN,FYN,SRC respectively with siRNA,knowing down of LYN BNDF didn' t regulate p-MLC2 phosphorylation.4.LYN could regulate MLCK phosphorylation levelLysates from HEK 293 cells overexpressing LYN-Myc(con group)or TrkB-Flag were immunoprecipitated(IP)with Myc antibody then incubating with protein A agarose beads and analyzed by immunobolting(IB),we found that LYN had interaction with MLCK.Then blocking down expression of LYN,the gray value of p-MLCK descended(p<0.05).Meanwhile overexpression of LYN,the gray value of p-MLCK increased.We found that BDNF could activate SRC signal and increase MLCK phosphorylation level leading to MLC2 phosphorylation by a series means of cytobiology and molecular biology.