The Relationships Between Structure And Function Of JWA Binds To α-tubulin

The most human diseases are the result of interactions between environmental factors and individial genetic susceptibility, but the mechanisms of the interactions are far to be understood. An increasing number of environmental responsive genes are cloned and found to be associated with human diseases. Some of environmental responsive genes are found involved in multi-signal transduction pathways and with a cross-talk manner to against the environmental stresses and keep the cell and body in homeostasis. JWA, a novel retinoic acid inducible and cytoskeleton associated gene, was primary cloned by Zhou et al (AF070523, 1998). The further evidences have shown in this research group that JWA seems a multi-functional gene. In one hand, it regulates cellular differentiations and apoptosis in multi tumor cell lines andprimary leukemia cells and associated with PKC signal pathway. In structurally, JWA protein binds with beta-tubulin and the both showed parallel distributions and could changed by treatment of physical and chemical microtubule targeting agents. In another hand, we found that JWA effectively involves in the regulation of intracellular amino acids transportation in PC 12 cells and this role might be associated with intracellular calcium signals. In this study, we focused on the relationships between the structure and function of JWA binds to alpha-tubulin. Firstly, we examined the dynamic changes in morphology of JWA and microtubule under disassembling agents treatment in PC 12 cells. Secondary, we designed series of models to investigate if JWA is also binds with alpha-tubulin and involves in cell cycle, especially in cell mitosis; and probe to observe the cellular localization of JWA in the PC 12 cells treated with environmental chemical podophyllotoxin.1. The relationships between JWA and a-tubulin in PC12 cellsWe previously confirmed that JWA protein and B-tubulin showed simultaneously and co-existed distribution patterns with treatment of antimitotic chemical colchicines. To understand the intracellular binding of JWA protein and a-tubulin by immunoprecipitation, the results showed that JWA protein was identified by either anti-JWA antibody or anti-a-tubulin antibody, with or without antimitotic chemical Nocodazole. JWA protein was unable detected by a non-specific IgG (Caspase-3 ip). These results suggested that JWA protein and a-tubulin are naturally intracellular binding each other and with a parallel distribution.Pretreatment of PC 12 cells on ice 2 hours (0C) to deploymerize the both proteins, and then put the treated cells back into 37C culture condition to recover them to original distribution patterns. The both proteins are still showed simultaneously and co-existed distributionpatterns during the processes. These results suggested that JWA protein and a-tubulin are polymerized and depolymerized simultaneously and with almost completely co-localization patterns. To clarify the influence of increased and decreased expression of JWA protein on the relationships between a-tubulin and JWA protein, we firstly successfully established JWA expression vector and was transfected HEK293 cells and then exposed on ice treatment for 2h. The both proteins showed depolymerized and co-existed distribution patterns. Secendly, after antisense-JWA oligonucleotide (5 X 10-5 mol.L-1) treatment for 48h and with colchicines (1 X 10"5 mol.L-1) treatment at different time periods, the expression of JWA protein was down-regulated; the depolymerization rate of both JWA protein and a-tubulin was increased when compared with the control . These results suggested that JWA protein stably bound to a-tubulin no matter it was at a higher or lower expressing level; however, the lower expression of JWA protein might speed up the depolymerizing rate of a-tubulin. Therefore, JWA might be an effective regulator of the a-tubulin polymerization.2. JWA involves in cell mitosis and regulates microtubularstabilityIt is likely that JWA protein is also associated with cell cycle. We treated PC12 cells with 0.5% bovine serum for...