The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosomabrucei

This study reports the identification and characterization of the regulator y subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosom a brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N-terminal domain, the pseudosubstrate site involved in bin ding and inactivation of the catalytic subunit, and two C-terminally locate d, 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, a nd it is strongly inhibited by a synthetic protein kinase inhibitor peptide . Unexpectedly, this kinase activity could not be stimulated by CAMP, but b y cGMP only. Binding studies with recombinant cyclic nucleotide binding dom ains of TbRSU confirmed that both domains bind cGMP with K-d values in the lower micromolar range, and that up to a 100-fold excess of cAMP does not c ompete with cGMP binding.