Cloning And Immunological Function Analysis Of Amphip38 Gene In

The mitogen-activated protein kinases (MAPKs) are intracellular kinases responding to extracellular stimuli to regulate fundamental cellular processes. P38MAPKs is important member of the MAPK family, is considered to be participated in many signal transduction pathways. These MAPKs are activated by dual phosphorylation on the Thr-Xaa-Tyr motif. P38 are activated by the upstream MAPK kinases MKK3 and MKK6 via dual phosphorylation of the Thr-Gly-Tyr motif in the activation loop. The activation of p38 MAP kinase can result in phosphorylation of transcription factor, as well as downstream kinases and then regulate the cell survival, cell cycle, and apoptosis. p38 MAP kinases was found to play important roles in the immune response, pro-inflammatory and cellular stresses response. Amphioxus, as transitional species from invertebrates to vertebrates in evolutionary history, belongs to the ancient chordate lineage. Amphioxus is a crucial organism for the understanding of chordate evolution including the origin of p38 MAPKs pathway and the immune system. In this study, based on the clone of p38, real time PCR, western bolting, we study the Branchiostoma belcheri p38 gene and the results are as follows:(1) We cloned the full-length of Amphip38 (Branchiostoma belcheri) gene cDNA through RACE. Three fragments were assembled to form a 1329bp nucleotide sequence. The full length Amphip38 cDNA contains a 1110bp open reading frame (ORF), a 88bp 5’-UTR and a 131bp 3’-UTR. The ORF encoded a putative 42kDa protein with 369 amino acids and the theoretical PI (isoelectric point) of 5.52.The Amphip38 protein contains a STK (serine/threonine kinase) domain that was conservative domain of p38 family proteins.(2) From homology analysis of the Amphip38 protein sequence with other known p38 MAP kinases, we found that the deduced amino acid sequence of Amphip38 has high identity (80.5-84%), and similarity (67.2-72.5%) to the other known p38 MAP kinases homologs. The Amphip38 protein had the highest identity (72.5%) to Dicentrarchus labrax MAPK14. Which indicate that Amphip38 we cloned is homology gene of vertebrate p38.(3) Analysis of the putative protein sequence revealed that it had a conserved serine/threonine kinase domain (STK, residues 29-313aa). In which, there were the TGY motif, the CD motif, the glycine-rich (GxGxxG) ATP binding loop, ED site, and the substrate binding site. They are important for the interaction of p38 MAP kinase with substrate, activators and regulators. We further predicted the 3D structure of Amphip38, the 3D structure of Amphip38 is similar to that of human p38a. There is a groove on the surface of p38 protein. The catalytic site (activation-loop) was inside the groove. Docking sites, CD and ED were also found in the 3D structure of amphip38. The differences in CD domain may be related to the different amino acid composition of the upstream and downstream.(4) p38 MAP kinase protein sequences from different animals were used to construct a phylogenetic tree. Evolutionary analysis demonstrated that Amphip38 gene was ancestor of vertebrate p38 gene, and the p38 gene had undergone gene duplication and/or differentiation in the evolutionary from invertebrate to vertebrate and that result in the generation of four isoforms (p38 α、 p38 β 、p38 γ和 p38δ)) of p38 MAP kinase family.(5) Based on the quantitative RT-PCR analysis, we found that the Amphip38 gene was ubiquitously expressed in tissues we tested, while expression levels varied among the different tissues:Amphip38 expression was abundant in notochord and muscle, and moderate in the hepatic cecum, low in intestine and gill. The highest expression in notochord and muscle may be related to the tissue-specific functions of Amphip38. The predominant expression in muscle and notochord may be involved in the regulation of cell differentiation. The high expression in hepatic cecum may relate to the inflammation response, as for hepatic cecum is the precursor of vertebrate liver.(6) The mRNA level of Amphip38 was measured at different time point (2 h,4 h, 6 h,8 h,12 h,24h) after LPS stimulation by quantitative RT-PCR. The relative expression of Amphip38 was up-regulated after LPS infection at 4 h, peaked at 6 h, and declined to the primary level at 8 h. The expression of Amphip38 is obvious increased after the LPS stimuli. The result indicated that Amphip38 might participate in the immune response.(7) We examined the phosphorylation-p38 MAP kinase (p-p38) in amphioxus upon LPS stimulation by Western bolting (0.5 h,1 h,3 h,6 h). It is obvious that p-p38 MAP kinase is increased at 0.5 h and 1 h, but decreased at 6 h post LPS treatment. These results verified that Amphip38 was involved in immune by dual phosphorylation of p38 MAP kinase.In conclusion, we cloned and functionally characterized Amphip38 gene for the first time. Our results indicate that Amphip38 is involved in mediating innate immune in amphioxus and is the ancestor gene of vertebrate p38 MAP kinase group. Our current study will provide valuable information to take an insight into molecular mechanism of innate immune and the evolution of vertebrate p38 MAP kinase family.