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Related Experiment Videos

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

R J Fisher1, S Koizumi, A Kondoh

  • 1Laboratory of Cellular Biochemistry, Program Resources, Inc./DynCorp, Frederick Cancer Research and Development Center, Maryland.

The Journal of Biological Chemistry
|September 5, 1992
PubMed
Summary

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Human ETS1 proteins, crucial in T-cell leukemia, bind DNA sequences. Their function is modulated by thiol group modifications, impacting protein interactions and cellular context.

Area of Science:

  • Molecular Biology
  • Oncogenes
  • Protein Biochemistry

Background:

  • The ETS1 proto-oncogene plays a role in cellular processes.
  • Understanding ETS1 protein function and regulation is vital in T-cell leukemia research.

Purpose of the Study:

  • To isolate and characterize human ETS1 proteins from T-cell leukemia lines.
  • To investigate the role of thiol (-SH) groups in ETS1 protein function and DNA binding.
  • To analyze the interaction of ETS1 with the PEA3 DNA motif.

Main Methods:

  • Immunoaffinity chromatography for ETS1 protein isolation.
  • NH2-terminal amino acid sequencing for identity confirmation.
  • Electrophoretic mobility shift assays (EMSA) to study DNA-protein complex formation.

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  • Zone sedimentation analysis to determine the binding stoichiometry.

    • Main Results:

    • Human ETS1 proteins were isolated and identified from CEM T-cell leukemia line.
    • ETS1 proteins exist in thiol (-SH)-available and -SH-protected states, influenced by N-ethylmaleimide (NEM) and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK).
    • ETS1 specifically binds the PEA3 (CAGGAAGT) DNA motif, forming a complex detectable by EMSA. Reduced ETS1 is necessary for complex formation, but thiol modification does not inhibit binding, although it alters complex mobility.

    Conclusions:

    • ETS1 protein function, particularly DNA binding, is sensitive to modifications of its thiol groups.
    • The monomeric form of ETS1 is responsible for binding to the PEA3 oligonucleotide.
    • These findings provide insights into the post-translational regulation of ETS1 activity in a cellular context.