Ca(2+) and calmodulin modulate DNA-binding activity of maize heat shock transcription factor in vitro.
DNA-binding activity of a maize heat shock transcription factor (HSF) was induced by heat shock of a whole cell extract at 44 degrees C. Addition of the calcium ion chelator EGTA reduced the binding of the HSF to heat shock element (HSE) in vitro. Re-addition of CaCl(2) to the sample pretreated with EGTA restored the ability of the HSF to bind to DNA. DNA-binding activity of the HSF was also induced by directly adding CaCl(2) to a whole cell extract at non-heat-shock temperature, but not by MgCl(2). During HS at 44 degrees C, calmodulin (CaM) antagonists chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) inhibited DNA-binding activity of the HSF in a concentration-dependent manner, but N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), an inactive structural analogue of W7, did not. Addition of antiserum specific to CaM reduced the binding of the HSF to HSE. Re-addition of CaM to the sample pretreated with antiserum could restore the binding activity of the HSF. DNA-binding activity of the HSF was promoted by directly adding CaM to a whole cell extract at 44 degrees C, but not by 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 BSA. Our observations further confirm the role of Ca(2+) in activation of the HSF in plant and provide the first example of the role of CaM in regulation of DNA-binding activity of the HSF. These results suggest that Ca(2+) and CaM are involved in HSP gene expression likely through regulating the activity of the HSF.
Li
Bing
B
Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang 050016, P.R. China.
Liu
Hong-Tao
HT
Sun
Da-Ye
DY
Zhou
Ren-Gang
RG
D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology; D010940:Plant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D002147:Calmodulin; D002614:Chelating Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D010940:Plant Proteins; D014157:Transcription Factors; D004247:DNA; D002122:Calcium Chloride; D002118:Calcium