The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei

Eur J Biochem. 2001 Dec;268(23):6197-206. doi: 10.1046/j.0014-2956.2001.02564.x.

Abstract

This study reports the identification and characterization of the regulatory subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosoma brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N-terminal domain, the pseudosubstrate site involved in binding and inactivation of the catalytic subunit, and two C-terminally located, closely spaced cyclic nucleotide binding domains. Immunoprecipitation of TbRSU coprecipitates a protein kinase activity with the characteristics of protein kinase A: it phosphorylates a protein kinase specific substrate, and it is strongly inhibited by a synthetic protein kinase inhibitor peptide. Unexpectedly, this kinase activity could not be stimulated by cAMP, but by cGMP only. Binding studies with recombinant cyclic nucleotide binding domains of TbRSU confirmed that both domains bind cGMP with Kd values in the lower micromolar range, and that up to a 100-fold excess of cAMP does not compete with cGMP binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cell Line
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / chemistry*
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cyclic GMP / metabolism
  • Cyclic GMP-Dependent Protein Kinases / chemistry*
  • Cyclic GMP-Dependent Protein Kinases / genetics
  • Cyclic GMP-Dependent Protein Kinases / metabolism
  • DNA, Protozoan / genetics
  • Drosophila
  • Genes, Protozoan
  • Kinetics
  • Molecular Sequence Data
  • Protein Subunits
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Transfection
  • Trypanosoma brucei brucei / enzymology*
  • Trypanosoma brucei brucei / genetics

Substances

  • DNA, Protozoan
  • Protein Subunits
  • RNA, Messenger
  • RNA, Protozoan
  • Recombinant Fusion Proteins
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclic GMP-Dependent Protein Kinases
  • Cyclic GMP