Identification and Characterization of Novel Mutations in the Human Gene Encoding the Catalytic Subunit Calpha of Protein Kinase A (PKA)

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser109 significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly186 completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly186 is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg2+, which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser109 or Gly186 may be faced with abnormal development and possibly severe disease

Pain modulators regulate the dynamics of PKA-RII phosphorylation in subgroups of sensory neurons

Knowledge about the molecular structure of protein kinase A (PKA) isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a high content screening microscopy approach, we identified the RIIβ subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIβ-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain-eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory-neuron-derived F11 cells that the inflammatory mediator PGE₂ specifically activated PKA-II but not PKA-I. Accordingly, pain-sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIβ as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.status: publishe

Pain modulators regulate the dynamics of PKA-RII phosphorylation in subgroups of sensory neurons.

Knowledge about the molecular structure of PKA isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a High Content Screening microscopy approach, we identified the RIIβ subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIβ-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory neuron-derived F11 cells that the inflammatory mediator PGE2 specifically activated PKA-II but not PKA-I. Accordingly, pain sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIβ as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state

Regulation of cAMP-dependent Protein Kinases: THE HUMAN PROTEIN KINASE X (PrKX) REVEALS THE ROLE OF THE CATALYTIC SUBUNIT αH-αI LOOP*

cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIβ but not by substrate inhibitors RIIα or RIIβ. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2–4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes

Primers used for sequencing of <i>PRKACA</i> exons 2 to 10.

<p>Primers used for sequencing of <i>PRKACA</i> exons 2 to 10.</p

Mutations in the <i>PRKACA</i> gene discovered by sequencing of 498 Norwegian subjects.

<p>Nucleotide enumeration is counted from the PKA Cα1 transcript start codon. Amino acid numbering is given for the mature protein with N-terminal Gly₁ corresponding to codon 2. Sequence conservation is illustrated in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838.s001" target="_blank">Fig. S1</a>.</p

Mutation of Gly186 in Cα1 prevents Cα from binding ATP and divalent cations.

<p><b> A.</b> The 3D structure of the catalytic site of Cα1. Selected conserved motifs and their relations to divalent cations Mn1 and Mn2 and ATP are shown. Residues connecting phospho-Thr₁₉₇ (pThr₁₉₇) to the DFG motif are represented as stick models <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838-TenEyck1" target="_blank">[50]</a>. Mn₂ATP (green), the DFG motif (teal), Gly-rich loop (salmon), catalytic loop (yellow), and activation loop (purple) are also highlighted. <b>B.</b> Overall structure of Cα1_WT with the conserved structural motifs the C- and R-spine structural motifs highlighted. The boxed segments depict spatial relations between residue 186 (Gly or Val) and ATP, divalent cations, and the C- and R-spines. <b>C.</b> DFG motif in Cα1_WT (left) and Cα1_Gly186Val (right) and its relations to Mn1 and ATP. Residues are represented as stick models with carbon (orange), oxygen (red) and nitrogen (blue) atoms. The hydrogen bond between the side chain of Asp₁₈₄ and the amide group of Gly₁₈₆ (dashed line) is predicted to be broken in Cα1_Gly186Val due to the Val side chain. The models are based on the structure with PDB identifier 3FJQ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838-Thompson1" target="_blank">[48]</a>.</p

Sequences of human wild type PKA Cα1/Cα2, location of point mutations, and activities of mutant proteins.

<p><b>A.</b> The human Cα wild type and mutated amino acid sequence. The N-terminal methionine of Cα1 is removed post-translationally, and glycine residue 1 in mature Cα1 corresponds to codon 2. Alternating black and blue coloring shows the contribution of the eleven exons to the mature translated protein. The codons for Val₁₅ and Ser₁₃₉ (red) are encoded by nucleotides from neighboring exons. The <i>PRKACA</i> gene encodes two splice variants, Cα1 and Cα2 (UniProt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838-TheUniProt1" target="_blank">[59]</a> accession number P17612), which differs by alternative use of exons 1–1 or 1–2, respectively. The four point mutations are highlighted and denoted with the amino acid change. The Gly186Val mutation is located in the DFG motif (boxed) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838-Taylor1" target="_blank">[26]</a>. <b>B.</b> The location of the four mutations are indicated (red) in a model of Cα1 based on the PDB structure 3FJQ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034838#pone.0034838-Thompson1" target="_blank">[48]</a>. Residues are designated according to the WT Cα1 sequence. ATP and two divalent cations are shown in yellow. <b>C.</b> Immunoblotting and phosphotransferase assays of WT and mutated Cα1 subunits. Cα1 was expressed in HEK 293T cells and immunoreactive C subunit detected with anti-C (Anti-mouse PKA[C], cat. no. 610981) after separation of cell extracts by SDS-PAGE in 10% gels and immunoblotting. Expression of Cα1_WT is shown in lane 1 (WT), mutated Cα1 in lanes 2 to 5, and endogenous Cα1 in lane 6 (Mock). Activities are normalized relative to Cα1_WT activity. Data represents mean values ± standard deviation (SD) of triplicate experiments (*, P < 0.05. **, P < 0.005).</p