The Functional Studies Of Domains And Phosphorylation Sites Of SLP76

Our early research found that there are 12 proteins interact with intracellular domain of receptor of advanced glycation end products(RAGE) in human lung cDNA library by T7 phAGEs display,and SH2 domain-containing leukocyte protein of 76kD(SLP76) is one of them.The experiment to identify the interaction between SLP76 and RAGE in vivo and in vitro also confirm the interaction,and it is stimulation dependent.SLP76 is a hematopoietic cell-specific adaptor protein that is crucial for T-cell receptor (TCR) signaling,hemostasis and platelet function.SLP76 can interact with several proteins in cell and has the ability to involve in many signal transduction pathways.SLP76 contains an amino-terminal SAM domain,a carboxyl-terminal SH2 domain and several phosphorylation spots and rich proline between them.Advanced glycation end products(AGEs) are nonenzymatical adducts of proteins,lipids, and nucleic acids which form in a time-dependent manner in a pro-oxidant environment, especially when target molecules turnover slowly,such as aging,and some diseases such as diabetes or atherosclerosis.Different from other receptors,the AGEs-RAGE interaction mediate downstream signal transduction pathways other than discard of AGEs.As both of RAGE and SLP76 can active nuclear factor NF-κB,and they also have the interaction.We can have a hypothesis that when the ligands interact with RAGE,the introcelullar domain of RAGE is associated with adaptor protein complex composited by SLP76 directly or indirectly.Grb2 which is a part of the complex leads SLP76 to the Ras.Then Ras can active MAPKKK(Raf,etc.),and the signal transduct by a highly conserved cascade consisting of three kinds of kinases(MKKK/MKK/MAPK) result in generation of cellular oxidant stress and activation of NF-kB.Finally,the expression spectrum of cytokine will change.If the hypothesis is true,it would make great sense to the functional study of the interaction of RAGE and SLP76 in signal transduction,and we could link the interaction of RAGE and SLP76 and pathogenesis of the diseases above together.In order to study the factions of SLP76 we constructioned a series of mutants of it.Then our experiment contains the follow five steps.First step,the preparation of AGEs.Second step, the construction of mutants of SLP76.Third step,identify the effects of mutants of SLP76 on MAPK.Fourth step,identify the effects of mutants of SLP76 on it's tyrosine phosphorylation. The last step,identify the effects of mutants of SLP76 on RAGE-SLP76 interaction. To sum up,we have successfully prepared AGEs with biological activity and constructe mutants of SLP76 which have stable expression in HEK 293 cell line.Our immunoprecipitation experiments initially identified the domains which necessary for RAGE-SLP76 interaction,they are SAM domain,tyrosine on the 145 and 173 sites and serine on the 207 sites.Furthermore we certificate that the mutants of SLP76 have no impact on the phosphorylation of ERK,p38 and it's tyrosine phosphorylation under the stimulate with AGEs.