The Effect Of Calcium Ions-calmodulin Signal System On Regulation Of Heat Shock Transcription Factor Activity

Our previous work has shown that Ca2+-CaM signal system is involved in HS gene expression. Herein, we mainly focus on the mechanism by which Ca2+-CaM plays a role at the level of HSF. Maize was used as a main experimental material.Gel mobility shift assay was employed to study the relationship between Ca2+-CaM and DNA-binding activity of heat shock transcription factor (HSF). The binding activity of the HSF to heat shock element (HSE) was induced by heat shock (44 ℃) of a maize whole cell extract. DNA-binding activity of the HSF increased in a time-dependent manner, it increased gradually with time of HS and reached its maximum after 30 min of HS. Competition binding experiment indicates that the binding of the HSF to synthetic HSE is specific. Under HS at 44℃, addition of the calcium ion chelator EGTA to a whole cell extract reduced the binding of the HSF to HSE. Re-addition of CaCl2 to the sample pretreated by EGTA restored the binding activity of the HSF to HSE obviously. DNA-binding activity of the HSF was also induced by directly adding Ca2+ to a whole cell extract at non-heat-shock temperature, but not by the same concentration of MgCl2. Effect of 1mM CaCl2 is most prominent. During HS at 44 ℃, the CaM antagonists W7 and CPZ also inhibited DNA-binding activity of the HSF in a concentration-dependent manner, but not by W5, an inactive structural analogue of W7. Addition of antiserum specific to CaM to a whole cell extract reduced binding of the HSF to HSE at 44℃. Re-addition of CaM to the cell extract pretreated with antiserum might restore the ability of the HSF to bind to HSE. DNA-binding activity of the HSF was promoted by directly adding CaM to a whole cell extract at 44 ℃, but not by the same concentration of S100 and BSA. Moreover, at non-heat-shock temperature, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract but not by the same concentration of BSA. Our observations further confirm the role of Ca2+ in activation of HSF in plant and provide the first example of the role of CaM in regulation of the DNA-binding activity of the HSF. These results suggest thatCa2+ and CaM were involved in HS genes expression likely thlough regulating the activity of HSRSome studies have suggested that HSP70 is a negative regulatory factor. It represses activity of HSF by formation of complex with HSF under non-heat-shock condition. The result of co-immunoprecipetation showed that HSF-HSP70 complex existed in cytoplasmic extract. Gel mobility shift assay was used to study the affect of HSP70 on the activity of HSF. These results provide evidence for negative regulation role of HSP70 in plant.Our previous work has also shown that there is a CaM-binding site within maize cytoplasmic HSP70 and that HSP70 binds CaM in a Ca2+-dependent manner (Sun et al, 2000). The high conservation of CaM-binding suquence implies that the binding of CaM to HSP70 might have an important biological function. It was shown that CaM-HSP70 complex existed in heat shocked cytoplasmic extract by co-immunoprecipetation experiment. The result indicates that CaM regulates HS gene expression likely through binding to HSP70.