Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation.
Cys(2)His(2) zinc finger proteins make up the largest class of transcription factors encoded in the genomes of higher eukaryotes. Recent studies of the Ikaros transcription factor demonstrated that this zinc finger protein undergoes cell cycle-dependent changes in association with DNA that seem to be due to phosphorylation of Thr or Ser residues in the linker regions connecting adjacent zinc finger domains. The high degree of conservation of this linker sequence within the Cys(2)His(2) superfamily suggested a common mechanism for the cell cycle-dependent modulation of DNA-binding affinity throughout this large class of transcription factors. The effects of linker phosphorylation on DNA-binding affinity were investigated through a direct comparison of the DNA-binding properties of four synthetic zinc finger proteins produced by native chemical ligation. The four proteins, comprising three zinc finger domains joined by two consensus Thr-Gly-Glu-Lys-Pro linkers, correspond to all four possible combinations of linker Thr phosphorylation states. Fluorescence-based DNA-binding studies of a specific DNA-binding site revealed that phosphorylation of a single linker reduced binding affinity approximately 40-fold, whereas phosphorylation of both linkers reduced binding affinity 130-fold. These results with purified components demonstrate that linker phosphorylation does, indeed, produce a significant reduction in DNA-binding affinity and support a model wherein a single cell cycle-dependent Ser/Thr kinase could simultaneously inactivate a large number of zinc finger transcription factors.
Jantz
Derek
D
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. bergj@mail.nih.gov
Berg
Jeremy M
JM
D000595:Amino Acid Sequence; D002245:Carbon Dioxide / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding
D010750:Phosphoproteins; D002245:Carbon Dioxide; D004247:DNA