Reversible phosphorylation of a protein from Trypanosoma equiperdum that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases

Abstract

Homologous proteins of the cAMP-dependent protein kinase (PKA) regulatory and catalytic subunits have been identified in Trypanosoma equiperdum (TeqR-like and TeqC-like, respectively). Partially purified TeqR-like from parasites isolated in the presence of glucose migrated as an apparent 55 kDa/57 kDa polypeptide doublet when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a single polypeptide of 57 kDa was obtained when parasites were deprived of glucose, a condition that has been shown to activate a TeqC-like enzyme. As revealed by immunoblots using anti-phospothreonine antibodies, the 57 kDa band corresponded to a form of TeqR-like that was phosphorylated in threonine residues. TeqR-like phosphorylation was reversible since the level of phospho-TeqR-like decreased once glucose was readded to glucose starved-parasites. Dephospho- and phospho-TeqR-like proteins are monomers with native molecular masses of 54.93-57.41 kDa, Stokes radii of 3.42-3.37 nm, and slightly asymmetric shapes (frictional ratio f/fo = 1.36-1.32). A protein kinase of ∼40 kDa was also partially purified from glucose deprived-trypanosomes, which corresponded to the TeqC-like enzyme by its ability to phosphorylate kemptide, its inhibition by PKA-specific inhibitors, and its immunorecognition by anti-PKA catalytic subunit antibodies. TeqR-like and TeqC-like did not coelute following anion-exchange chromatography, revealing that these proteins are not associated forming a complex in T. equiperdum. Yet, when TeqR-like was incubated in vitro with TeqC-like in the presence of Mg²⁺ and ATP, the 55 kDa dephospho form of the 55kDa/57 kDa polypeptide doublet of TeqR-like was converted into the 57 kDa phospho form, demonstrating that TeqR-like is a substrate for TeqC-like.

Keywords: Dourine; Protein phosphorylation; Regulatory and catalytic subunits of the cAMP-dependent protein kinase; Trypanosoma equiperdum; Trypanosomatid parasites.