Negative Control Of MEK/ERK Signaling By Spinophilin-Targered Synaptic PP1 In Spinal Cord Dorsal Horn

Objective: Peripheral tissue injury activates MEK/ERK signaling in spinal cord dorsal horn,which plays an essential role in nociceptive sensitization.Protein phosphatase-1(PP1)is one of the important serine/threnonine phosphatases.When targeted at excitatory glutamatergic synapses,PP1 is able to regulate the phosphorylation levels of synaptic substrates and involved in the control over synaptic efficacy and plasticity.Spinophilin(SPN)is an F-actin-and PP1-binding protein,which can direct PP1 to the specific postsynaptic compartments.The current study was designed to investigate the influence of SPN-targeted synaptic PP1 on MEK/ERK signaling and its significance in chronic inflammatory pain.Methods: We performed the coimmunoprecipitation and GST pull-down assays to examine the possible interaction between SPN and MEK/ERK.The effect of SPN-bound PP1 on MEK/ERK activities was investigated through in vitro dephosphorylation assays.Excitatory postsynaptic currents(EPSCs)were recorded in spinal cord lamina ? neurons to test the effect of SPN/PP1 complex on nociceptive synaptic transmission.We also induced inflammatory pain by interdermal injection of Complete Freund's Adjuvant(CFA)in rats,and evaluated the possible role of SPN/PP1 in chronic inflammatory pain.Results:1.SPN interacted with ERK and MEK,but not with Raf-1,in spinal cord dorsal horn.2.The PDZ domain located within the C-terminal region of SPN mediated the molecular interaction with MEK1 and ERK2.3.SPN-targeted synaptic PP1 dephosphorylated MEK/ERK and negatively controlled their enzymatic activities.4.MEK and ERK,once dephosphorylated by SPN-bound PP1,dissociated with SPN.5.Inteference with SPN/PP1 complex in spinal cord dorsal horn of intact rats induced the activation of MEK1/2 and ERK1/2,as evidenced by their significant phosphorylation.6.The regulatory effect of SPN-bound PP1 on MEK1/2 and ERK1/2 depended on the synaptic activity.7.Through MEK/EKR signaling,SPN-targeted synaptic PP1 tightly controlled the excitatory synaptic transmission mediated by N-methyl-D-aspartate subtype receptors(NMDAR).Viral expression of SPN(F451A)to disturb SPN/PP1 interaction in spinal dorsal horn neurons specifically enhanced the function of postsynaptic NMDAR,leading to the potentiation of NMDAR-mediated synaptic currents.SPN(F451A)didn't affect the synaptic transmission mediated by AMPA receptors.8.GluN2 B subunit-containing NMDAR at synapses served as the specific target for the regulation by MEK/ERK signaling initiated by SPN/PP1.Disruption of SPN/PP1 complex significantly enhanced the synaptic expression of GluN2 B receptors.9.ERK phosphorylation of MLCK might contribute to the regulatory effect of SPN-targeted PP1 on GluN2 B synaptic transmission.10.Peripheral inflammation was able to disturb SPN/PP1 association in spinal cord dorsal horn,thus removing the negative control by SPN/PP1 over MEK/ERK activities.11.Overexpression of SPN(F451A)mutant in spinal dorsal horn neurons of intact rats was sufficient to evoke ERK-and GluN2B-dependent pain sensitization.12.Intrathecal injection of recombinant adenovirus to infect wild-type SPN [SPN(WT)] in spinal dorsal horn of rats repressed MEK/ERK activities caused by peripheral inflammation,blunted GluN2 B receptor-mediated nociceptive synaptic transmission and effectively alleviated inflammatory hyperalgesia and allodynia.Conclusion: SPN-targeted synaptic PP1 was engaged in the nociceptive modification in spinal cord dorsal horn by meas of negative control of MEK/ERK signaling pathway.