Structural Basis for the Regulation of Protein Kinase A by Activation Loop Phosphorylation

• Published in: The Journal of biological chemistry 2012-04, Vol.287 (18), p.14672-14680
• Main Authors: Steichen, Jon M., Kuchinskas, Michael, Keshwani, Malik M., Yang, Jie, Adams, Joseph A., Taylor, Susan S.
• Format: Article
• Language: English
• Subjects: Activation Loop; Animals; Catalytic Domain; Crystal Structure; Crystallography, X-Ray; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Inactivation; Enzyme Kinetics; Hydrogen Bonding; Mice; Models, Molecular; Mutation, Missense; Phosphorylation; Phosphorylation - genetics; Protein Kinase A (PKA); Protein Structure and Folding; Protein Structure, Secondary; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.335091

The catalytic subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC group of protein kinases. Whereas PKA has served as a structural model for the protein kinase superfamily, all previous structures of the catalytic subunit contain a phosphorylated activation loop. To understand the structural effects of activation loop phosphorylation at Thr-197 we used a PKA mutant that does not autophosphorylate at Thr-197. The enzyme crystallized in the apo-state, and the structure was solved to 3.0 Å. The N-lobe is rotated by 18° relative to the wild-type apoenzyme, which illustrates that the enzyme likely exists in a wide range of conformations in solution due to the uncoupling of the N- and C-lobes. Several regions of the protein including the activation loop are disordered in the structure, and there are alternate main chain conformations for the magnesium positioning loop and catalytic loop causing a complete loss of hydrogen bonding between these two active site structural elements. These alterations are reflected in a 20-fold decrease in the apparent phosphoryl transfer rate as measured by pre-steady-state kinetic methods. Background: Activation loop phosphorylation is a conserved mechanism for regulating protein kinases. Results: The unphosphorylated C-subunit structure of protein kinase A shows decoupling of the two lobes of the enzyme. Conclusion: Phosphorylation orients the small and large lobes of the kinase for catalysis. Significance: PKA in its unphosphorylated state shows a great deal of structural disorganization, and this is difficult to predict in advance.