JWA, A Novel Microtubule Associated Protein, Regulates Homeostasis Of Intracellular Amino Acids In PC12 Cells

Human diseases are usually the results of interactions between environmental agents and genetics. There are an increasing number of environmental responsive genes to be found and related to human diseases. To investigate the interactions, we could elucidate the molecular mechanism of environmentally-induced diseases. In 1998, using the differential display high-throughput technique, Zhou et al cloned a novel environmental responsive gene from human bronchial epithelial (HBE) cells, named JWA, which is broadly distributed in most of examined eukaryotic cells and tissues. The intracellular distribution of the protein is very similar to that of cytoskeleton under immunofluorescence microscopy. The gene expressions are modulated by some cell differentiation inducers, such as, all trans retinoic acid (ATRA), 13-cis-retinoic acid and 12-O-tetradecanoyl-phorbol-13 acetate (TPA). Our recent studies showed that JWA is also a functionalprotein associated with cell differentiation and apoptosis, and might play an important role in development of acute promyelocytic leukemia (APL). It was reported that glutamate transporter EAAC1-associated protein (GTRAP3-18), expressed by E-18 which is 95% identical to JWA, is a novel protein that interacts with excitatory amino acids transporters 3 (EAAT3) and negatively modulates Na+-dependent glutamate transporter EAAT3-mediated glutamate uptake in rat. In this study, we firstly probed to observe the precise cellular localization of JWA in the HBE cells or NIH3T3 cells, the different localization of JWA during cell mitosis. Further more we examined the details of JWA and microtubule disassembly when cells were treated with physical, chemical factors. Secondary, confirming that JWA is a novel microtubule associated protein, we detected the variety of intracellular amino acids using knock down JWA in PC 12 cell culture model in order to study how JWA regulates homeostasis of intracellular amino acids.1. Study on the relation between JWA and microtubule.Tubulin is typically isolated by pelleting microtubules form tissues, depolymerizing the microtubules by cooling them to 4癈, centrifuging the cooled solution to remove the insoluble material, and then polymerizing the tubulin-containing supernatant by warming to 37癈 in the presence of GTP. Co-purification of JWA with tubulin suggested that it is not nonspecific contaminants but rather molecules that interact specifically with microtubules. Immunoprecipitation analysis also showed that JWA protein always could be recognized by both JWA- and p-tubulin-antibodies from NIH3T3 cells treated with or without depolymerizing reagent (20uM nocodazole). Using immunoelectronic microscopy technique, we also observed JWA is co-localized with p-tubulin. The laser confocal microscopy provided evidences that the JWA is intracellular localized and dense around thenucleolus relatively reaching the cytomembrane in radical directions. Double-labeled (FITC/rhodamine) immunofluorescence microscopy images showed JWA parallels that of microtubules. In JWA transfected HBE cells, we observed very similar responses of both JWA and P-tubulin to the treatments of depolymerizing reagents colchicine and nocodazole. However, it seemed that JWA is less sensitive to cold-shock (4C, 2 hrs) treatment than that of P-tubulin in HBE cells. But we failed to repeat this result in NIH3T3 cells although the time-coursed depolymerizing model was successfully repeated. The expression of JWA protein in NIH3T3 cells is significantly blocked but has no obvious affect on its cytoplasmic distribution by the treatment of antisense-JWA-oligonucleotide (ajo, 10uM, 24 hrs). Interphase NIH3T3 cells show the intracellular distributions of microtubules parallel that of JWA. The mitotic cells, however, show that JWA protein locates around spindles, much like a spindle packing protein. The detailed evidences needed to be further investigated.2. JWA regulates the homeostasis of intracellular amino acidsSix antisense-JWA-oligonucleotides (ajos), 15bp each, were corresponding to...