A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators.
A novel CaM-binding protein was isolated through protein-protein interaction based screening of an Arabidopsis cDNA expression library using a 35S calmodulin (CaM) probe. There are four additional homologs in the Arabidopsis genome with similar structures: a BTB domain in the N-terminus and a Zf-TAZ domain in the C-terminus. Hence, they were designated as AtBT1-5 (Arabidopsis thaliana BTB and TAZ domain protein). CaM-binding experiments revealed that all five AtBTs are CaM-binding proteins, and their CaM-binding domains were mapped to the C-terminus. AtBT homologs are also present in rice, but are not present in human, animal, yeast or other organisms, suggesting that the BTB and TAZ domain proteins are plant-specific. The AtBT1-smGFP fusion protein expressed in tobacco BY-2 cells showed that AtBT1 targets the nucleus. Yeast two-hybrid screening using an AtBT1 fragment as bait identified two interacting proteins (AtBET10 and AtBET9) belonging to the family of fsh/Ring3 class transcription regulators. The BTB domain of the AtBTs is required for the interaction, and this protein-protein interaction was confirmed by GST pull-down. AtBET10 also interacts with AtBT2 and AtBT4, and exhibited a transcriptional activation function in yeast cells. AtBTs exhibit varying responses to different stress stimuli, but all five genes responded rapidly to H2O2 and salicylic acid (SA) treatments. These results suggest that AtBTs play a role in transcriptional regulation, and signal molecules such as Ca2+, H2O2, and SA affect transcriptional machinery by altering the expression and conformation of AtBTs which interact with transcriptional activators such as AtBET10.
Center for Integrated Biotechnology and Department of Horticulture, Washington State University, Pullman, WA 99164-6414, USA.
Liqun
L
Du
B W
BW
Poovaiah
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D029681:Arabidopsis Proteins; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D020156:Salicylic Acid; D002118:Calcium