Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

Regulation of Membrane Targeting of the G Protein-coupled Receptor Kinase 2 by Protein Kinase A and Its Anchoring Protein AKAP79

The beta 2 adrenergic receptor (beta 2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta 2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta 2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta 2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta 2AR markedly inhibit phosphorylation of beta 2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbeta gamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta 2AR and phosphorylation of agonist-occupied beta 2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization

Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation

Tauroursodeoxycholic acid exerts anticholestatic effects by a cooperative cPKC alpha-/PKA-dependent mechanism in rat liver.

Objective: Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in part by protein kinase C (PKC)-dependent mechanisms. Its taurine conjugate, TUDCA, is a cPKCa agonist. We tested whether protein kinase A (PKA) might contribute to the anticholestatic action of TUDCA via cooperative cPKCa-/PKA-dependent mechanisms in taurolithocholic acid (TLCA)-induced cholestasis. Methods: In perfused rat liver, bile flow was determined gravimetrically, organic anion secretion spectrophotometrically, lactate dehydrogenase (LDH) release enzymatically, cAMP response-element binding protein (CREB) phosphorylation by immunoblotting, and cAMP by immunoassay. PKC/PKA inhibitors were tested radiochemically. In vitro phosphorylation of the conjugate export pump, Mrp2/Abcc2, was studied in rat hepatocytes and human Hep-G2 hepatoma cells. Results: In livers treated with TLCA (10 mmol/l)+TUDCA (25 mmol/l), combined inhibition of cPKC by the cPKCselective inhibitor Go¨6976 (100 nmol/l) or the nonselective PKC inhibitor staurosporine (10 nmol/l) and of PKA by H89 (100 nmol/l) reduced bile flow by 36% (p,0.05) and 48% (p,0.01), and secretion of the Mrp2/ Abcc2 substrate, 2,4-dinitrophenyl-S-glutathione, by 31% (p,0.05) and 41% (p,0.01), respectively; bile flow was unaffected in control livers or livers treated with TUDCA only or TLCA+taurocholic acid. Inhibition of cPKC or PKA alone did not affect the anticholestatic action of TUDCA. Hepatic cAMP levels and CREB phosphorylation as readout of PKA activity were unaffected by the bile acids tested, suggesting a permissive effect of PKA for the anticholestatic action of TUDCA. Rat and human hepatocellular Mrp2 were phosphorylated by phorbol ester pretreatment and recombinant cPKCa, nPKCe, and PKA, respectively, in a staurosporine-sensitive manner. Conclusion: UDCA conjugates exert their anticholestatic action in bile acid-induced cholestasis in part via cooperative post-translational cPKCa-/PKA-dependent mechanisms. Hepatocellular Mrp2 may be one target of bile acid-induced kinase activation

Gravin orchestrates protein kinase A and 2-adrenergic receptor signaling critical for synaptic plasticity and memory

A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of protein kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D (phosphodiesterase 4D) to the β2-adrenergic receptor. Here, we show that mice lacking the α-isoform of gravin have deficits in PKA-dependent long-lasting forms of hippocampal synaptic plasticity including β2-adrenergic receptor-mediated plasticity, and selective impairments of long-term memory storage. Furthermore, both hippocampal β2-adrenergic receptor phosphorylation by PKA, and learning-induced activation of ERK in the CA1 region of the hippocampus are attenuated in mice lacking gravin-α. We conclude that gravin compartmentalizes a significant pool of PKA that regulates learning-induced β2-adrenergic receptor signaling and ERK activation in the hippocampus in vivo, thereby organizing molecular interactions between glutamatergic and noradrenergic signaling pathways for long-lasting synaptic plasticity, and memory storage

Characterization of Freshwater Natural Dissolved Organic Matter (DOM): Mechanistic Explanations for Protective Effects Against Metaltoxicity and Direct Effects on Organisms

Dissolved organic matter (DOM) exerts direct and indirect influences on aquatic organisms. In order to better understand how DOM causes these effects, potentiometric titration was carried out for a wide range of autochthonous and terrigenous freshwater DOM isolates. The isolates were previously characterized by absorbance and fluorescence spectroscopy. Proton binding constants (pKa) were grouped into three classes:acidic (pKa ≤ 5), intermediate (5 \u3c pKa ≤ 8.5) and basic (pKa \u3e 8.5). Generally, the proton site densities (LT) showed maximum peaks at the acidic and basic ends around pKa values of 3.5 and 10, respectively. More variably positioned peaks occurred in the intermediate pKa range. The acid–base titrations revealed the dominance of carboxylic and phenolic ligands with a trend for more autochthonous sources to have higher total LT. A summary parameter, referred to as the Proton Binding Index (PBI), was introduced to summarize chemical reactivity of DOMs based on the data of pKa and LT. Then, the already published spectroscopic data were explored and the specific absorbance coefficient at 340 nm (i.e. SAC340), an index of DOM aromaticity,was found to exhibit a strong correlation with PBI. Thus, the tendencies observed in the literature that darker organic matter is more protective against metal toxicity and more effective in altering physiological processes in aquatic organisms can now be rationalized on a basis of chemical reactivity to protons

Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of β2-adrenergic receptor (β2AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric β2ARs that undergo endocytosis, whereas PKA modifies dimeric β2ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated β2ARs are enriched in dendrites, whereas GRK-phosphorylated β2ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated β2ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell

Benchmarking pKa prediction

Background: pKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data. Results: Here we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs. Conclusion: Overall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures