Global consequences of activation loop phosphorylation on protein kinase A

Abstract

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 Thr(197) in the activation loop when expressed in Escherichia coli. Although mutation of Arg(194) to Ala prevents autophosphorylation, phosphorylation of Thr(197) 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 Thr(197) decreased the K(m) by approximately 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 alphaH-alphaI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu(208) and Arg(280).

FIGURE 1.

Interactions of the activation loop phosphate in the C-subunit and other conserved regions of the activation segment. The activation segment is colored olive with the sequence shown below the structure along with two common consensus sequences of PKA substrates. Phe¹⁸⁵ and Leu⁹⁵ are part of the hydrophobic regulatory spine (4) and are shown as a tan molecular surface. ATP is colored black. The P-3 site and position of Arg¹⁹⁴ are in bold, and the P-site is colored red.

FIGURE 2.

Phosphorylation state of R194A mutants. R194A mutants, with and without treatment by PDK1 or wild type C-subunit, were probed with α-Thr(P)¹⁹⁷, α-Ser(P)³³⁸, and α-C-subunit antibody.

FIGURE 3.

Fluorescence polarization of FLU-IP20 binding to the C-subunit. Titration of WT and mutant C-subunits (R194A and pR194A) was at fixed concentrations of ATP and fluorescently labeled IP20. The values are the means ± S.E., n = 3 for R194A and pR194A and n = 2 for WT.

FIGURE 4.

Urea-induced unfolding profiles. A, tryptophan fluorescence of the Wild type C-subunit is shown at varying concentrations of urea. B, comparison of the urea denaturation curves for WT, R194A, and pR194A C-subunit. The fraction of protein unfolded is shown as a function of urea concentration monitored by intrinsic tryptophan fluorescence.

FIGURE 5.

H/DMS data for peptides with increased rate of deuterium exchange in the unphosphorylated mutant. Time course for deuterium exchange in the small lobe with peptides 1 (residues 41–54), 2 (residues 55–59), and 3 (residues 109–116) and in the active site region, peptides 4 (residues 163–172) and 5 (residues 182–186) shown in red. The scale of the y axis is the maximum number of exchangeable amides. The large lobe of the C-subunit (residues 127–350) is colored olive, and the small lobe (residues 1–126) is colored gray for reference.

FIGURE 6.

Deuterium exchange in the activation segment and the αH-αI loop. Time course for deuterium exchange in the large lobe with peptides 1 (residues 199–211) and 2 (residues 278–296) colored in red. The scale of the y axis is the maximum number of exchangeable amides. The large lobe of the C-subunit (residues 127–350) is colored olive, and the small lobe (residues 1–126) is colored gray for reference.

FIGURE 7.

Stable helical core of the PKA catalytic subunit. The percentages of deuterium exchange after 50 min in deuterium in peptides from helices C, E, F, H, and J are shown. WT (white bars) and R194A (black bars). Helical peptides that exchanged less than 10% of their amide hydrogens are mapped onto the structure of the C-subunit and colored blue. The A-Helix is a fast exchanging helix and is shown in red. Where bars are not shown, no exchange was observed. The large lobe (residues 127–350) is colored olive, and the small lobe (residues 1–126) is colored gray.