The Bayesian inference trees for vertebrate PKA Cα and Cβ both closely reflects the evolutionary relationships among these organisms.

<p><b>A</b> Phylogenetic analysis of Cα orthologs resulted in a tree that was rooted with human and mouse Cβ as outgroups. The tree was based on the nucleotide sequences of exons 2 to 8 (all codon positions, GTR+Γ+I model). <b>B</b> Phylogenetic analysis of Cβ orthologs was performed employing nucleotide sequence data (all codon positions, exons 2 to 10, GTR+Γ+I model). The resulting tree was rooted with human and mouse Cα as outgroups. In both trees, branch lengths are shown as substitutions per site, with scale indicated by the scale bars. Bayesian posterior probabilities are given for each node and ML bootstrap values (1000 replications) are shown for selected nodes where the clades are identical in the Bayesian and ML analysis. In addition to organisms found in Fig. 2, representative sequences from the following species were included: eutherian mammals rhesus macaque (<i>M. mulatta</i>), tarsier (<i>T. syrichta</i>), dog (<i>C. familiaris</i>), horse (<i>E. caballus</i>), pig (<i>S. scrofa</i>), cow (<i>B. taurus</i>), rat (<i>R. norvegicus</i>), and hamster (<i>C. griseus</i>), marsupial mammals wallaby (<i>M. eugenii</i>) and opossum (<i>M. domestica</i>), the frog <i>X. laevis</i>, the pufferfish <i>T. nigroviridis</i> and Atlantic salmon (<i>S. salar</i>). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a> for the sequence data.</p

The identity of eleven amino acids in the protein chain may define the Cα and Cβ branches of PKA catalytic subunits.

<p>Our full set of PKA catalytic subunits (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a>) from bony fishes and tetrapods, comprising 27 Cα and 33 Cβ, was employed to identify eleven amino acid positions that together may be used to classify a PKA catalytic subunit as belonging to one of the two branches. The sequence logos define the PKA Cα and Cβ clades within the <i>Teleostomi</i>, which includes the familiar classes of bony fishes, birds, mammals, reptiles, and amphibians. We find invariable Gln35, Thr37, Glu64, Gly66, His68, Ser109 and Glu334 in Cα and invariable Asp42, Gln67, and Arg319 in Cβ (Cα1/Cβ1 numbering). The residues in the corresponding positions in human Cα1 and Cβ1 are also shown.</p

Phylogenetic relationships among the PKA catalytic subunit homologs in chordates.

<p>The Cα and Cβ paralogs are a result of a gene duplication in a common ancestor of vertebrates. Subsequent duplications of Cα and Cβ in a teleost fish ancestor have resulted in four PKA catalytic subunits in these organisms. The Bayesian inference tree is based on the nucleotide sequences (codon positions 1 and 2 only, GTR+Γ+I model) of exons 2 to 10 which corresponds to a multiple sequence alignment with no gaps. The phylogram is shown with estimated branch lengths proportional to the number of substitutions at each site, as indicated by the scale bar. The arthropod fruit fly (<i>D. melanogaster</i>) and the echinoderm sea urchin (<i>S. purpuratus</i>) have been set as outgroups. Bayesian posterior probabilities are shown for each node. The topology of a maximum likelihood (ML) tree generated with the same data set and model was identical to the Bayesian inference tree. ML bootstrap values are shown for selected nodes (1000 replications). The sequences of human and mouse PKA Cα and Cβ and the homologs from amphioxus (<i>B. floridae</i>), zebra finch (<i>T. guttata</i>), chicken (<i>G. gallus</i>), the frog <i>X. tropicalis</i>, the lizard <i>A. carolinensis</i>, medaka (<i>O. latipes</i>), the pufferfish <i>T. rubripes</i>, and stickleback (<i>G. aculeatus</i>) are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060935#pone.0060935.s003" target="_blank">Materials and Methods S1</a>. The <i>X. tropicalis</i> Cα and <i>A. carolinensis</i> Cβ are incorrectly placed (See discussion and Fig. 3).</p

Activity of wild type and Gly186Val mutated Cα1 and Cα2.

<p><b>A.</b> Phosphotransferase activity of Cα1_WT and Cα1_Gly186Val expressed in HEK-293T cells was measured in the presence of Kemptide and γ-³²P-ATP, and in the presence (+) and absence (−) of cAMP or PKI kinase inhibitor. Enzyme activity was normalized according to activity of Cα1_WT which was set to 100%. The data are presented as mean values ± SD of triplicate experiments. <b>B.</b> Recombinant Cα2_WT (not shown) and Cα2_Gly186Val were expressed in BL21 (DE3) cells. For Cα2_Gly186Val, expressed protein was purified by running cell extracts over a Ni-resin loaded with His6RIIα_Gly337Glu affinity column and eluted with 10 mM cAMP. The purification steps of Cα2_Gly186Val are shown after separation of the various fractions by SDS-PAGE. The crude cell extract (lane 1, crude) was centrifuged and divided into a soluble (lane 2) and an insoluble fraction (lane 3, pellet). Proteins not retained on the column is shown (lane 5, Flowthrough). W1, W3, W4 and W5 (lanes 6 and 8–10) depict protein contents of successive washing steps using buffer containing 50 mM NaH₂PO₄ (pH 8.0), 5 mM β-mercaptoethanol, and 25 mM KCl. E1-E4 (lanes 11–14 ) depict protein content in consecutive elution fractions using buffer containing 50 mM NaH₂PO₄ (pH 8,0), 10 mM cAMP, 5 mM β-mercaptoethanol, and 25 mM KCl. To assure equal amounts of RIIα_Gly337Glu and Cα2_Gly186Val, cell extracts of RIIα_Gly337Glu and Cα2_Gly186Val expressed in separate bacteria cultures were mixed 1∶1 (lane 7, 1∶1 mix). Protein with the correct molecular mass (arrow to the right) was obtained in fractions E1–E3. Ten µl was applied in lanes 6, 8, 9 and 10, seven µl in lane 1, 2, 3, 5 and 7, and five µl in lane 4. (Molecular Weight Marker (MWM)). <b>C.</b> Kinase activity of recombinant Cα2_WT and Cα2_Gly186Val was determined by employing the spectrophotometric Cook assay. The data are presented as mean values ± SD of triplicate experiments.</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

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

Primers for mutagenesis (mutated nucleotide in bold).

<p>Primers for mutagenesis (mutated nucleotide in bold).</p