cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.

We characterized the ontogeny of cAMP-dependent protein kinase (PKA) enzymatic activity and PKA subunit mRNA expression in developing lung. The lungs of fetal Sprague-Dawley rat pups were removed after 16, 18, or 20 days of gestation and at term. PKA activity was greatest in the 18- and 20-day gestation lungs. Tissue cAMP levels were lowest in the 16-day lungs and increased with lung maturity. We were able to detect only low levels of mRNA for the C beta subunit of PKA by northern blot analysis of total lung RNA and we were able to detect mRNA for the RI beta and RII beta subunits only by RT-PCR. Therefore, we limited our analysis of PKA subunit mRNA levels to those for C alpha, RI alpha and RII alpha. The mRNA levels for C alpha, were highest in the 16-day lung, decreased at 18 and 20 days, were lower in the newborn and lowest in the adult lung. RI alpha mRNA levels were also highest at 16 days and lowest in the adult lung. However, RII alpha mRNA levels were similar in the 18-day, 20-day and newborn lungs. Dexamethasone treatment of fetal lung explants resulted in a small decrease in RI alpha mRNA levels but was not associated with a change in PKA activity. We conclude that PKA activity and PKA subunit mRNA expression are developmentally regulated in fetal lung. Such regulation results in optimal PKA activity at the time of type II alveolar cell differentiation, presumably in preparation for air breathing. The absence of an effect of glucocorticoid on PKA activity suggests that glucocorticoids are not responsible for the increase in PKA activity which accompanies this critical time in lung maturation.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals.

We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells.

Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the β-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to β-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).

Stimulation of beta-adrenergic receptors activates type I and II cyclic AMP-dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase-anchoring proteins (AKAPs) regulates cyclic AMP-dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus alpha-actinin-specific antibodies or AKAP100 plus ryanodine receptor-specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.

Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.

The interaction of intermediate filaments (IFs) with the cell-cell adhesion complexes desmosomes is crucial for cytoskeletal organization and cell resilience in the epidermis and heart. The intracellular desmosomal protein desmoplakin anchors IFs to the cell adhesion complexes predominantly via its four last carboxy-terminal domains (C-terminus). However, it remains unclear why the C-terminus of desmoplakin interacts with different IF types or if there are different binding affinities for each type of IFs that may influence the stability of cell-specific adhesion complexes. By yeast three-hybrid and fluorescence binding assays, we found that the coiled-coil 1 of the conserved central rod domain of the heterodimeric cytokeratins (Ks) 5 and 14 (K5/K14) was required for their interaction with the C-terminus of desmoplakin, while their unique amino head- and C-tail domains were dispensable. Similar findings were obtained in vitro with K1/K10, and the type III IF proteins desmin and vimentin. Binding assays testing the C-terminus of desmoplakin with assembled K5/K14 and desmin IFs yielded an apparent affinity in the nM range. Our findings reveal that the same conserved domain of IF proteins binds to the C-terminus of desmoplakin, which may help explain the previously reported broad binding IF-specificity to desmoplakin. Our data suggest that desmoplakin high-affinity binding to diverse IF proteins ensures robust linkages of IF cytoskeleton and desmosomes that maintain the structural integrity of cellular adhesion complexes. In summary, our results give new insights into the molecular basis of the IF-desmosome association.