Abstract
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.
Highlights
Activation loop phosphorylation is a conserved mechanism for regulating protein kinases
Other AGC kinases such as PDK1, co-crystallized with ATP, show only subtle structural changes even though the kinase is catalytically inactive without activation loop phosphorylation [8]
By crystallizing the R194A mutant of the protein kinase (PKA) C-subunit we were able to compare the structural consequences of removing this single phosphate from the activation loop without altering the rest of the protein, including Thr-197 and the phosphorylated Ser-338
Summary
Activation loop phosphorylation is a conserved mechanism for regulating 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. At the N terminus of the activation loop there is a conserved DFG motif, called the magnesium positioning loop because the aspartate coordinates magnesium at the active site This region has been found to be a common regulatory motif in protein kinases. Inactivation occurs when this phenylalanine moves out of the hydrophobic pocket, disrupting the orientation of the DFG aspartate and in some cases sterically blocking the ATP binding site [4, 5] These are referred to as “DFG-out” conformations. Due to its proximity to the activation loop phosphorylation site, the DFG phenylalanine is the key regulatory element of the hydrophobic R-spine that bridges the N- and C-lobes of the kinase. The C-spine is a noncontiguous hydrophobic ensemble that traverses both lobes of the enzyme and is completed by the adenine ring of ATP [9]
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