Screen And Characterize The Interacting Proteins Of Bak1-Specific Mutants

BAK1 is a receptor kinase with diverse functions in Arabidopsis,mediating multi signal pathways including BR signal,innate immunity,cell death,as well as plant growth and development.Kinase activity and phosphorylation-sites study would be beneficial to further reveal the molecular mechanisms that BAK1 interacted with other proteins to transduce the downstream signaling.In this study,intracellular kinase domain of BAK1-flag-WT(W),kinase inactivity BAK1-flag-K317E(E)and specific mutants BAK1-flag-A/D(A/D)were prokaryotic expressed in vitro,of which the kinase activity were further studied;And we screened the specific components among all the identified interaction components between BAK1(W/E)and specific mutants(A/D)through the MBP pull-down assay.Otherwise,we detected the phosphorylation level of BAK1 transgenetic plants in vivo.Finally,the tomato SIBAK1 was analyzed in bioinformatic.The main results are as follows:1.BAK1 kinase activity analysis.(1)The kinase activity and protein modification of W and A,which were prokaryotic expressed in vitro,varied at different inducing temperatures.W and A were strongly autophosphorylated and occurred an unknown modification at 25 ℃ or 30 ℃,then the SDS-PAGE gel showed characteristic double bands.While W and A showed a significant difference at 37 ℃,W showed the double bands which was severely phosphorylated,and A showed only the bottom band.D performed similarly with the kinase inactive E,they showed a unique band and completely lacked the phosphorylated upper band at designed inducing temperature;(2)The kinase domain of BAK1 that prokaryotic expressed caused the growth suppression of host cells,which was closely related to the kinase activity.It showed that the colony size of W was decreased significantly,while A,D and E showed no difference when compared to normal bacterials at 0.01 mM IPTG Interestingly,when IPTG concentration at 0.10 mM,the bacterial strains of W and A were failed to growth;and the growth of D and E were suppressed and their colony size became smaller.The results were corresponding to the previous study that BAK1 regulated the cell death negatively in vivo which was associated with the kinase activity.2.Screen and characterize the interacting components of BAK1-specific mutants.(1)The protein binding ability varied at different kinds of BAK1-specific mutants induced at distinct temperature.W that induced at 30 ℃ or 37 ℃ could bind PUB13-MBP,FLS2-MBP,PP2A-MBP,PEPR1/2-MBP,Atlg09640-MBP,BRI1-MBP,GSTF10-MBP,GST-MBP,and the W-binding capacity with Atlg09640-MBP,AtGSTF10-MBP,GST-MBP were rather weak.A induced at 30 ℃ can interact with all of those components,while no interaction can be detected among all the components except for PUB13-MBP,BRI1-MBP which showed strong interaction.(2)The protein modification affected the protein-protein interaction.The differences were mainly reflected in the bands that detected by Western Blot.Some of proteins such as FLS2-MBP,PP2A-MBP,PEPR1/2-MBP,BRI1-MBP interacted with the modificated W and A,namely,the phosphorylated upper band were detected by Western Blot.Some of proteins such as PUB13-MBP,Atlg09640-MBP,GSTF10-MBP,GST-MBP interacted with could detected the double bands,suggesting that both modificated and unmodificated W had the binding capacity with those proteins.While some of proteins such as PUB13-MBP,Atlg09640-MBP,GSTF10-MBP,GST-MBP only interacted with unmodificated A and showed the bottom band,briefly,the protein modification played an important role in peotein interaction.3.The phosphorylation level detection of BAK1 transgenic plants in vivoThe BAK1 overpression transgenic plants were cultuted in liquid medium,and the 11 days seedings were treated with flg22,Epi-BL,H2O2,NaCl,Basta,respectively,followed by immunoprecipitation and phosphorylation detection via Western Blot.The results suggested that the flg22 and Epi-BL treatments affected the phosphorylation level of BAK1,indicating that the phosphorylation level of BAK1 is closely related to multiple factors.4.The bioinformatics analysis of SLBAK1The analysis of amino acid sequences and protein structure indicated that SLBAK1 is highly homologous with AtBAK1,and belongs to the LRR receptor kinase family.The same kinase domain structure suggested that they would have similar biological functions.