Reduction in DNA-binding affinity of Cys ₂ His ₂ zinc finger proteins by linker phosphorylation

Abstract

The Cys 2 His 2 zinc finger proteins represent one of the largest classes of proteins found in the genomes of all eukaryotes that have been sequenced to date (1, 2). These proteins typically have multidomain architectures in which individual zinc-binding domains approximating the consensus sequence (Tyr,Phe)-XCys-X 2,4 -Cys-X 3 -Phe-X 5 -Leu-X 2 -His-X 3-5 -His are joined into arrays of two or more by relatively short linker sequences. As this protein class initially came to be characterized, it was observed that the linker sequence connecting adjacent zinc finger domains tends to be remarkably highly conserved. This sequence typically deviates only slightly from the consensus His-Thr-Gly-Glu-LysPro-(Tyr,Phe)-X-Cys. Indeed, the conservation of this sequence allowed cDNAs encoding many zinc finger proteins to be identified before genomic sequencing efforts (3). Although structural studies (4-7) and mutagenesis experiments (8-12) have suggested a role for the linker in DNA binding, it has been noted that this contribution is relatively minor and probably insufficient to explain the high degree of evolutionary conservation of the linker sequence (10).Recently, an attractive hypothesis explaining linker sequence conservation was proposed based on the observation that the Cys 2 His 2 zinc finger protein Ikaros was excluded from mitotic chromosomes (13). Ikaros protein isolated from mitotic cell extracts was found to be phosphorylated and seemed to have reduced DNA-binding affinity. The sites of phosphorylation were localized to residues within the linkers connecting the protein's four zinc finger domains. The linkers in murine Ikaros have the sequences Thr-Gly-Glu-Arg-Pro, Ser-Gly-Glu-Lys-Pro, and Ser-Val-Gly-Lys-Pro, approximating the Thr-Gly-Glu-LysPro consensus sequence (phosphorylation residues are in italics). Replacement of the Thr and Ser residues in these sequences with Ala resulted in a loss-of-protein phosphorylation, suggesting that these residues are the sites of modification. Based on these observations, it was proposed that the linker sequence acts as the substrate for an unidentified cellular kinase capable of phosphorylating Cys 2 His 2 zinc finger proteins in a cell cycledependent manner. It has long been known that transcription ceases upon entry into mitosis and that this phenomenon is due, in part, to the mitotic inactivation of gene-specific transcription factors (14). The proposal that a cell cycle-dependent kinase can phosphorylate the Thr-Gly-Glu-Lys-Pro linker sequence provides a plausible explanation for the linker's high degree of evolutionary conservation while at the same time offering a mechanism for the mitotic inactivation of a large number of sequence-specific transcription factors.Because the previous report was limited to semipurified components from whole cell extracts, it was impossible for the authors to evaluate the effects of linker phosphorylation on DNA-binding affinity in a quantitative manner. To test the hypothesis that phosphorylation of linker sequences is responsible fo...

Motif Decomposition of the Phosphotyrosine Proteome Reveals a New N-terminal Binding Motif for SHIP2

“…Recently a role for phosphorylation of zf-C 2 H 2 domains in inhibition of transcription has been suggested (43,44), supposedly as a consequence of the negatively charged phosphomoiety that reduces DNA affinity (45). Indirectly our results support this; because we did not retrieve any interaction partner for the synthesized phosphopeptide matching the zinc finger motif, it is unlikely that this motif directs proteinprotein interaction, but rather phosphorylation of this motif modulates nucleic acid binding.…”

Regulation of the Transcription Factor YY1 in Mitosis through Phosphorylation of Its DNA-binding Domain

“…Additional support of this conclusion was shown in an in vitro biochemical study that demonstrated that addition of a phosphate group to the linker peptides of zinc finger proteins results in a dramatic reduction in DNA-binding affinity (Jantz and Berg, 2004). This mode of inactivation is not a unique to YY1.…”

Quantitative 3D elemental microtomography ofCyclotella meneghinianaat 400-nm resolution

“…Elevated Zn concentrations within the cytoplasmic pillar may indicate the presence of Zn finger proteins. While such proteins are typically closely associated with nuclear material and are often found in association with nuclei in freshwater diatoms (6), they dissociate from the DNA prior to and during mitosis (13). Multiple smaller P-rich bodies, which possibly represent polyphosphate bodies or local aggregations of ribosomes containing nucleic acids, are apparent when the isosurface threshold is reduced (also visible in Fig.…”