Hepatitis B Virus X Protein Regulate Sprouty Homolog 1 Activation Of MAPK Signaling Pathway

The hepatitis B virus (HBV) X protein (HBx) plays an important role in HBV replication. HBx is a multifunctional protein that regulates numerous cellular signal transduction pathways, including modulating apoptosis and proliferation. It is mainly collaborative some key regulatory protein regulating cell signal transduction pathways, these regulation roles include activating host cell and the virus itself to gene transcription, cell apoptosis, inhibiting repair damaged DNA. Moreover, HBx activate of mitogen-activated protein kinases (MAPK),JAK,STAT the cell signal transduction pathways cascade of reactions. However, HBx involved in numerous signal transduction pathways, we are concerned about that the HBx activates the MAPK signal transduction pathway, because the pathway is a highly conservative module, and there are a variety of cell related to functions including cell proliferation, differentiation and migration, especially playing a significant regulatory role in the process of tumor formation and migration. In this regard no direct interaction has been found between HBx and any of these protein kinases. But the detailed molecular mechanism of the action of HBx has not been elucidated. Through studies on HBx and SPRY1, we describe the detailed molecular mechanism underlying and show HBx regulation SPRY1 that it is inhibition of MAPK receptor to activate the MAPK signaling pathway. In order to clarify the mechanism, the subject adopted a series of experimental studies:(1) Construction Flag-HBx,Myc-SPRY1,Myc-SPRYl-53-Mut eukaryotic expression vector ;(2) Research whether exist interaction between Flag-HBx and Myc-SPRY1; (3) Research interaction domain between Flag-HBx and Myc-SPRY1; (4) Whether Myc-SPRY1 would affect the Flag-HBx level of expression in HEK293 mammalian cells; (5) It will be researched between Flag-HBx and Myc-SPRY1 interaction on AP-1 transcription factor affect by AP-1 luciferase reporter gene experiments;(6) Research on HEK293 cell apoptosis and proliferation function affect between Flag-HBx and Myc-SPRY1 interaction. The conclusions are as follows:1. Successfully constructed Flag-HBx,Myc-SPRY1,Myc-SPRY1-53-Mut eukaryotic expression vector.2. By co-immunoprecipitation (Co-IP) and Pull-down experiments determine to the existence interaction between Flag-HBx and Myc-SPRY1.3. By co-immunoprecipitation experiments show that SPRY1 No.53 tyrosine mutations did not affect the their interaction, but the AP-1 luciferase reporter gene experiments show that SPRY1 No.53 tyrosine mutations affect AP-1 cell signal transduction pathway activation,we speculate that they may be the interaction region including a section of this sequence;4. Myc-SPRY1 proteins affect the Flag-HBx protein expression levels in the mammalian HEK293 cells with Myc-SPRY1 proteins the expression level increases;5. Flag-HBx collaborates Myc-SPRY1 activating AP-1-luc transcriptional activity.6. It was found that Flag-HBx inhibits the proliferation of HEK293 cells and promotes apoptosis of HEK293 cells. Myc-SPRY1 increased Flag-HBx inhibitory apoptosis and promoted proliferation the level of HEK293 cells when Myc-SPRY1 and Flag-HBx were co-transfected with HEK293 cells.