Identification Of Functional Phosphorylation Sites Of The Cytoplasmic Domain Of Rage

Advanced glycation end products (AGE) are nonenzymatical adducts of proteins, lipids, and nucleic acids which begin with linkage between thecarbonyl group from glucose or carbonyl compounds and the amino group to form Schiff bases and then Amadori compounds. The formation and accumulation of AGE are most accelerated under diabetes, AGE are ligands for receptor for AGE (RAGE) in the signal transduction pathway which play an important role in the complication of diabetes. At the meantime, AGE accumulation also occurs in euglycemia and aging, driven by oxidant stress and inflammation. One mechanism by which AGE might affect cellular function is by binding to the receptor for AGE (RAGE).Receptor for AGE (RAGE) is a multi-ligand receptor member of the immunoglobulin superfamily of cell surface molecules. Although RAGE was first identified as a signal transduction receptor for AGE, it also binds S100/calgranulin, amphoterin /HMGB-1 and amyloid. Different interactions of ligands with RAGE can cause cellular dysfunction in a number of pathophysiologically relevant situations such as diabetes, inflammatory, tumor cell behavior, amyloidosis, Alzheimer's disease, and also involved in the neuronal development. The interaction of these ligands to RAGE does not accelerate clearance or degradation but rather activates a sustained subsequence signal transduction molecules such as reactive oxygen species (ROS), Ras, mitogen-activated protein kinase (MAPK) and NF-κB pathway. Blockade of RAGE by F(ab')₂ fragments of antibodies prevent the activation of ERK1/2.However, the precise molecular mechanisms for the initiation of cell signal transduction by RAGE remain to be elucidated. RAGE has a highly charged intracellular domain of 41 amimo acids, it is possible that the intracellular domain plays a key role in the signal transduction pathway after stimulation.Because the reversible phosphorylation of proteins is one of the most important post-translational modifications and is also involved in a host of cellular processes. The phosphorylation of proteins plays a key role in the interaction between two proteins. It is possible that the activation of subsequence molecules by AGE may depend on the phosphorylation of RAGE, so identify the functional phosphorylation sites of RAGE is very important in determining the detailed mechanism of the RAGE signal pathway.According to two principles, one is the phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues, the other is the functional phosphorylation site of protein is conservative in the evolution of biology. To identify the functional phosphorylation sites of RAGE, we turned to the bioinformatic methods and the free bioinformatic sources online. After the primary structures among several species were carefully studied, some conservative amino acid residues were selected. Next, we chose four sites S391, S399, S400 and T401 as the candidate phosphorylation sites of RAGE.Then we successfully made the corresponding mutation constructs, pcDNA3-HA-RAGE(A391), pcDNA3-HA-RAGE(A399), pcDNA3-HA-RAGE(A400), pcDNA3-HA-RAGE(A401), in order to mutants S391, S399, S400 and T401 into alanine (A). In order to avoid the influence of endogenous RAGE, we choose a cell line which does not express RAGE (HEK 293). Firstly, we have to detect the phosphorylation of RAGE after the cell stimulated by AGE. After pcDNA3-HA -RAGE(WT) was transfected into HEK 293 cells, the cells were activated by AGE for different times, the cells were lysed and the supernatant was used in the immunoprecipitations with HA beads, and then through serine phosphorylation detection kit to detecte the phosphorylation of RAGE. The results demonstrated that under physiological condition, RAGE was serine phosphorylated, while the serine phosphorylation of RAGE markedly decreased after cell stimulation by AGE in comparison to the unstimulated cells. Secondly, we have to identify the exact phosphorylation sites of RAGE, we transfected pcDNA3-HA-RAGE(A391), pcDNA3-HA-RAGE(A399), pcDNA3-HA-RAGE(A400), pcDNA3-HA-RAGE (A401) into HEK 293 cells, pcDNA3-HA-RAGE(WT) was also transfected as a control. The cells were also lysed and the supernatant was used in the immunoprecipitations with HA beads, and then through phosphoserine detection kit and phosphothreonine detection kit to detect the phosphorylation of RAGE. The result showed that the serine phosphorylation level of the HA-RAGE(A400) was decreased, while the phosphorylation level of HA-RAGE(A391), HA-RAGE(A399), HA-RAGE(A401) was the same as RAGE(WT). It is proved that the S400 of RAGE is the phosphorylation site of RAGE. It is well known that ERK1/2, NF-κB can be activated by AGE in monocytes and tumor cells, and then a variety of cytokines (IL-1, IL-8, MCP-1, TNF, VEGF) are released. Therefore, we transfected different RAGE mutants into HEK 293 cells, then detect the phosphorylation level of ERK1/2 stimulated by AGE to further prove that the functional phosphorylation site of RAGE is the 400th serine of RAGE. After the transfected cells were activated by AGE for different duration, then the cells were lysed and the supernatant was used in Western-blot through phospho-ERK1/2 antibody. Our results showed that after transfected pcDNA3-HA-RAGE into HEK 293 cells and activated by AGE, the phosphorylation level of ERK1/2 was increased and the highest point was 5 mins. pcDNA3-HA-RAGE and each mutant was transfected into HEK 293 cells, after the cells were activated by AGE for 5 mins, then the cells were lysed and the supernatant was used in Western-blot through phospho-ERKl/2 antibody. Our results showed that after AGE stimulation HA-RAGE(A391), HA-RAGE(A399), HA-RAGE(A401) showed the same ability to activate ERK1/2 as the wild type while HA-RAGE(A400) lost this ability. This result suggested that the 400th serine of RAGE plays an important role in the downstream of the AGE-RAGE signal pathway and also affect the phosphorylation of ERK1/2. It is definitely proved that the 400th serine of RAGE is the functional phosphorylation site of RAGE.Through these studies, we draw the following conclusions:1. Under physiological condition, RAGE is serine-phosphorylated, while the serine phosphorylation of RAGE is markedly decreased after the cell stimulated by AGE in comparison to the unstimulated cells.2. After transfected pcDNA3-HA-RAGE(WT) into HEK 293 cells and stimulated by AGE, ERK1/2 is phosphorylated.3. The phosphorylation of ERK1/2 activated by AGE depends on the phsphorylation of the 400th serine of RAGE.4. S400 is a functional phosphorylation site of the cytoplasmic domain of RAGE.