Characterization of the cAMP-dependent protein kinase catalytic subunit Cγ expressed and purified from sf9 cells

Abstract

The Cγ and Cα subunits of the cAMP-dependent protein kinase (PKA) contain 350 amino acids that are highly homologous (83% amino acid sequence), with 91% homology within the catalytic domain (a.a. 40–300). Unlike Cγ, the Cα subunit has been readily purified and characterized as a recombinant protein in vitro, in intact cells, and in vivo. This report describes for the first time the expression, purification, and characterization of Cγ. The expression of active Cγ was eukaryote-specific, from mammalian and insect cells, but not bacteria. Active recombinant Cγ was optimally expressed and purified to homogeneity from Sf9 cells with a 273-fold increase in specific activity and a 21% recovery after sequential CM-Sepharose and Sephacryl S-300 chromatography. The specific activity of pure Cγ was 0.31 and 0.81 U/mg with kemptide and histone as substrates, respectively. Physical characterization showed Cγ had a lower apparent molecular weight and Stokes radii than Cα, suggesting differences in tertiary structures. Steady-state kinetics demonstrated that like Cα and Cβ, Cγ phosphorylates substrates requiring basic amino acids at P − 3 and P − 2. However, Cγ generally exhibited a lower K m and V max than Cα for peptide substrates tested. Cγ also exhibited a distinct pseudosubstrate specificity showing inhibition by homogeneous preparations of RIα and RIIα-subunits, but not by pure recombinant protein kinase inhibitors PKIα and PKIβ, PKA-specific inhibitors. These studies suggest that Cγ and Cα exhibit differences in structure and function in vitro, supporting the hypothesis that functionally different C-subunit isozymes could diversify and/or fine-tune cAMP signal transduction downstream of PKA activation.