| Rice,as sessile plants,need to face the constantly changing external environment,among which there are bad environments that harm the growth and development of plants.In response to these hazards,plants have evolved different defense mechanisms,in which phosphorylation and dephosphorylation of proteins play a key role.Rice receptor-like cytokinase LRRK1 is a kind of receptor-like kinase without extracellular domain.A rice 14-3-3 protein Gf14f was identified by mass spectrometry analysis of the LRRK1 complex extracted from IP in rice in early laboratory.In plants,14-3-3proteins,as bridge proteins,are widely involved in plant physiological and biochemical processes.Therefore,in order to reveal the specific molecular mechanism of LRRK1 and Gf14f,this study analyzed the interaction of the two proteins through molecular and biochemical experiments,and identified their phosphorylation sites.Specific research results are as follows:(1)Bioinformatics analysis showed that OsGF14f was expressed in all rice tissues,but the expression level was relatively high in hypocotyls,roots and leaves.GF14f contains abundant phosphorylation sites,and it is speculated that LRRK1 and GF14f may interact with each other,and GF14f is the substrate protein of LRRK1.(2)The results of the subcellular localization experiment showed that GF14f was localized in the cytoplasm.Three independent experiments,including yeast twohybridization,GST pull-down and BIFC,proved that LRRK1 and GF14f directly interact with each other,and the interaction site was located at the cell membrane.(3)The prokaryotic expression and purification of LRRK1 and GF14f were incubated in the phosphorylation reaction system in vitro.Western blot analysis with the phosphorylated antibodies showed that the serine phosphorylation signal of GF14 F was significantly enhanced in the experimental group with ATP compared with the control group without ATP.This indicates that LRRK1 phosphorylates GF14f.(4)Phosphorylatd of GF14f was analyzed by mass spectrometry,and a total of 9phosphorylation sites were identified,including 3 threonine sites: T224,T235 and T238,and 6 serine sites: S78,S100,S112,S143,S160 and S239.(5)The related phosphorylation site of GF14f was mutated into alanine to simulate dephosphorylation.Detected by phosphorylation antibody,the phosphorylation signal of GF14f was significantly decreased when S78 of GF14 F was mutated into alanine in vitro,indicating that S78 was the main phosphorylation site for LRRK1 phosphorylation of GF14f.We hypothesized that LRRK1 may complete the cascade transduction of related signals through phosphorylation of S78 site of Gf14f.In conclusion,this study proved that rice receptor-like cytokinase LRRK1 can interact with Gf14f,a member of the rice 14-3-3 protein family,and it was preliminarily found that LRRK1 mainly phosphorylates S78 of Gf14f,suggesting that LRRK1 may phosphorylate S78 of Gf14f to complete the phosphorylation signal transmission.These results provide a preliminary basis for further analysis of the signal transduction mechanism involved in rice LRRK1 and 14-3-3 proteins. |
