Direct Involvement of the Cyclic Nucleotide Binding Sites in the Cyclic-Nucleotide-Induced Charge Shift of Protein Kinases

  • Lynn Wolfe
  • Terry A. Woodford
  • Sharron H. Francis
  • Jackie D. Corbin
Part of the NATO ASI Series book series (volume 44)


The holoenzymes of cAMP- and cGMP-dependent protein kinases (cAK and cGK) exist as cyclic nucleotide-free and -bound forms (C.E. Cobb, A.H. Beth, and J.D. Corbin (1987) J. Biol. Chem. 262, 16566–74; L. Wolfe, S.H. Francis, L.R. Landiss, and J.D. Corbin (1987) J. Biol. Chem. 262, 16906–13), which can be resolved by DEAE chromatography due to a cyclic nucleotide-induced increase in surface electronegativity. In the present study, the isolated type I and type II regulatory subunits (RI and RII) of cAK also exhibited an electronegative charge-shift in the presence of cAMP, implying that the catalytic subunit was not involved in the apparent change in conformation resulting in the charge-shift of the holoenzyme. A mutant bovine RI that contained the cAMP binding sites, but was missing regions involved in dimerization and interaction with the catalytic subunit, was expressed in E. coli and purified. This truncated RI also demonstrated an electronegative charge-shift following cAMP binding, indicating that the binding sites themselves were directly involved in the shift. The increase in surface electronegativity of the cyclic nucleotide binding sites may either be a reflection of an overall conformational change due to cyclic nucleotide binding or it could be directly involved in regulating function.


High Performance Liquid Chromatography Dependent Protein Kinase Saturated Ammonium Sulfate High Performance Liquid Chromatography Column Cyclic Phosphate 
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Copyright information

© Springer-Verlag Berlin Heidelberg 1990

Authors and Affiliations

  • Lynn Wolfe
    • 1
  • Terry A. Woodford
    • 2
  • Sharron H. Francis
    • 1
    • 2
  • Jackie D. Corbin
    • 1
    • 2
  1. 1.Department of Molecular Physiology and BiophysicsVanderbilt UniversityNashvilleUSA
  2. 2.Howard Hughes Medical InstituteVanderbilt UniversityNashvilleUSA

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