TAILIEUCHUNG - Báo cáo Y học: The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei

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. | Eur. J. Biochem. 268 6197-6206 2001 FEBS 2001 The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei Tarek Shalaby Matthias Liniger and Thomas Seebeck Institute of Cell Biology University of Bern Switzerland 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. Keywords sleeping sickness protein kinase A African trypanosomes cyclic nucleotide signalling. The concept of cellular signaling by cyclic AMP cAMP has been maintained throughout evolution from bacteria to mammals. However the only component of this signalling pathway that has been strictly conserved is the second messenger molecule itself cAMP while the enzymatic machinery that generates and transduces the signal exhibits great variety. This is exemplified by the adenylyl cyclases which have developed into many different molecular structures 1-3 although their function is invariably to convert ATP to cAMP. A similarly wide range of structure and sequence diversity of functionally similar enzymes is found within the cAMP-specific phosphodiesterases PDEs . On the .

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