Authors

Discussion

cAMP-dependent protein kinase (PKA) has served as a prototype of the family of protein kinases. Three genes that encode for protein kinases related to the catalytic subunits of mammalian PKAs, namely PKAC1, PKAC2 and PKAC3, have been identified in the genome of various trypanosomatids ("http://tritrypdb.org"). In agreement with these findings, we are reporting a PKA catalytic subunit-like protein from Trypanosoma equiperdum, which enzymatic activity was prompted upon nutritional stress driven by glucose fasting. The kinase enzymatic activity of the parasite protein was measured with an electrophoretic gel-shift assay that employs a fluorescently modified synthetic heptapeptide known as kemptide (sequence: LRRASLG), which is a specific substrate for PKA and PKA-like enzymes. The parasite enzyme was capable of phosphorylating histone type II-AS, the synthetic peptide SP20 (sequence: TTYADFIASGRTGRRNSIHD), and the α isoform of the PKA type II regulatory subunit (RIIα-subunit), which are known to function as substrates for the PKA catalytic subunit. Additionally, the ATP:phosphotransferase activity of the T. equiperdum protein kinase was inhibited by both the PKA-specific heat-stable peptide inhibitor PKI-α and the synthetic peptide IP20 (sequence: TTYADFIASGRTGRRNAIHD), which is derived from PKI-α. Surprisingly, the kinase enzymatic activity of the T. equiperdum PKA was independent of cAMP. In the present work, a methodology based on the formation of hybrid holoenzymes has been employed to purify the T. equiperdum PKA catalytic subunit-like protein from parasites that have been expanded in rats. By using polyhistidine-tagged mammalian PKA regulatory subunits that have been immobilized on a Ni²⁺-chelating affinity resin, stable heterologous holoenzyme complexes can be regenerated between the mammalian PKA regulatory subunits and the trypanosome protein kinase enzyme. Increasing the concentration of cAMP in the elution buffer resulted in the release and subsequent purification of the T. equiperdum PKA catalytic subunit-like protein.