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Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
11:24

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Published on: March 25, 2015

Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation

Derek Jantz1, Jeremy M Berg

  • 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. bergj@mail.nih.gov

Proceedings of the National Academy of Sciences of the United States of America
|May 7, 2004
PubMed

Insights

Phosphorylation of linker regions in Cys(2)His(2) zinc finger proteins significantly reduces their DNA-binding affinity. This finding suggests a common mechanism for cell cycle-dependent regulation of these important transcription factors.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Cys(2)His(2) zinc finger proteins are the largest class of transcription factors in higher eukaryotes.
  • The Ikaros transcription factor exhibits cell cycle-dependent DNA binding, potentially due to phosphorylation of linker regions.
  • Conserved linker sequences suggest a common regulatory mechanism across the Cys(2)His(2) superfamily.

Purpose of the Study:

  • To investigate the impact of linker phosphorylation on the DNA-binding affinity of Cys(2)His(2) zinc finger proteins.
  • To determine if phosphorylation of linker regions affects DNA-binding affinity in a dose-dependent manner.
  • To explore a potential common mechanism for cell cycle-dependent regulation of transcription factors.

Main Methods:

  • Synthesis of four distinct zinc finger proteins with all possible linker phosphorylation states using native chemical ligation.
  • Fluorescence-based DNA-binding assays to quantify binding affinity to a specific DNA site.
  • Comparative analysis of DNA-binding properties across different phosphorylation states.

Main Results:

  • Phosphorylation of a single linker region reduced DNA-binding affinity by approximately 40-fold.
  • Phosphorylation of both linker regions decreased DNA-binding affinity by approximately 130-fold.
  • These effects were observed using purified synthetic proteins.

Conclusions:

  • Linker phosphorylation significantly reduces DNA-binding affinity in Cys(2)His(2) zinc finger proteins.
  • A single cell cycle-dependent kinase could potentially regulate numerous zinc finger transcription factors simultaneously through linker phosphorylation.
  • This provides a mechanistic insight into cell cycle control of gene expression.

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