Protein phosphorylation induced by CCK_B-receptor in mouse neurons / 中国病理生理杂志

ABSTRACT

AIM:To explore the intracellular signaling transduction pathways in mouse neurons stimulated by octapeptide cholecystokinin(CCK8)on the base of phosphoproteome analysis and to evaluate the types of receptor by the use of CCK8 receptor specific antagonists.METHODS:Cultured neurons were divided into CCK stimulation group,CCK receptor antagonist group and control group at random.All of them were labeled with [32P] orthophosphate(2.03?108 Bq/L)for 2 h in 5% CO2 at 37 ℃.Control and stimulation groups were stimulated with DMEM and CCK8(10-7 mol/L)respectively for 60 min.The neurons in the antagonist groups were pre-incubated for 10 min with different type of receptor antagonists(10-8 mol/L),and then stimulated with CCK8(10-7 mol/L)for 60 min.Reactions were terminated by freezing neurons in liquid nitrogen,and then neuron proteins were collected.The [32P]-labeled lysates isoelectrically focused on IPG Drystrip pH4-7 linear gels and subsequently separated by second-dimensional SDS-PAGE.The 2-DE patterns of phosphoproteins were imaged with autoradiography for 7 days at-70 ℃.Then the phosphoprotein spots were inquired in Swiss-Prot and PPDB protein databases,and analyzed by PDQuest 2D software.RESULTS:Autoradiograph of the 2-DE maps showed that phosphorylation modifications of 46 proteins were involved in CCK signaling transduction in neuron,including protein kinase such as MK07,PI3K,PKC(?,?,?),PKA?,PKG?,AKT3 etc.Intracellular signal molecular such as RB17,GBO?,transcription factor,receptor for growth factor and nuclear protein were also observed.When the neurons were treated with L364 718,the phosphorylation of PKC? and P55G declined obviously,while the phosphorylation of PKC?,PKG?,OGFR and EGFR reduced when the CCK_B specific antagonist L365 260 was used.CONCLUSION:Both CCKA and CCK_B lead to the signaling transduction of CCK8 in neuron,but CCK_B probably play a more important role.The results suggest that cAMP-PKA,MAPK,JNK,PI3K-PKB and cGMP-PKG pathways are involved in CCK_B mediated signaling transduction.