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

Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.

Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10–100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925–949 and 1,140–1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3