title: The calmodulin-binding protein kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana.
abstract: Based on our previous findings, we proposed a pathway for the participation of Ca(2+)/calmodulin (CaM) in heat-shock (HS) signal transduction. The specific mechanism by which CaM regulates activation of heat-shock transcription factors (HSFs) is not known. CaM-binding protein kinases (CBK) are the most poorly understood of the CaM target proteins in plants. In this study, using a yeast two-hybrid assay, we found that AtCBK3 interacts with AtHSFA1a. Fluorescence resonance energy transfer was used to confirm the interaction between AtCBK3-YFP and AtHSFA1a-CFP. Furthermore, we demonstrate that purified recombinant AtCBK3 phosphorylated recombinant AtHSFA1a in vitro. We also describe the results of both downregulation of AtCBK3 expression and ectopic overexpression in Arabidopsis thaliana. The T-DNA insertion AtCBK3 knockout lines had impaired basal thermotolerance, which could be complemented by transformation of plants with the native gene. Overexpression of AtCBK3 resulted in plants with increased basal thermotolerance. Results from real-time quantitative PCR and protein gel-blot analyses suggest that AtCBK3 regulates transcription of heat-shock protein (HSP) genes and synthesis of HSPs. The binding activity of HSF to the heat-shock element (HSE), the mRNA level of HSP genes and synthesis of HSPs were upregulated in AtCBK3-overexpressing lines after HS, but downregulated in AtCBK3 null lines. These results indicate that AtCBK3 controls the binding activity of HSFs to HSEs by phosphorylation of AtHSFA1a, and is an important component of the HS signal transduction pathway.
authors.affiliation: Institute of Genetics and Physiology, Hebei Academy of Agricultural Sciences, Shijiazhuang 050051, China.
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Liu
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Li
authors.forename: Jin-Long
authors.initials: JL
authors.lastname: Han
authors.forename: De-Long
authors.initials: DL
authors.lastname: Liu
authors.forename: Da-Ye
authors.initials: DY
authors.lastname: Sun
authors.forename: Ren-Gang
authors.initials: RG
authors.lastname: Zhou
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005816:Genetic Complementation Test; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D006358:Hot Temperature; D016254:Mutagenesis, Insertional; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D010940:Plant Proteins; C036483:T-DNA; D014157:Transcription Factors; D017871:Calcium-Calmodulin-Dependent Protein Kinases