Global Consequences of Activation Loop Phosphorylation on Protein Kinase A

• Published in: The Journal of biological chemistry Vol. 285; no. 6; pp. 3825 - 3832
• Main Authors: Steichen, Jon M, Iyer, Ganesh H, Li, Sheng, Saldanha, S. Adrian, Deal, Michael S, Woods, Virgil L. Jr, Taylor, Susan S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 05.02.2010
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution; Catalysis; Catalytic Domain - genetics; Circular Dichroism; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Deuterium Exchange Measurement; Enzyme Activation; Enzyme Catalysis and Regulation; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Oligopeptides - metabolism; Phosphorylation; Protein Denaturation; Protein Folding - drug effects; Protein Structure, Tertiary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Threonine - metabolism; Urea - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.061820

Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr¹⁹⁷ in the activation loop when expressed in Escherichia coli. Although mutation of Arg¹⁹⁴ to Ala prevents autophosphorylation, phosphorylation of Thr¹⁹⁷ can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr¹⁹⁷ decreased the Km by ~15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the αH-αI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu²⁰⁸ and Arg²⁸⁰.