Impaired alveolar liquid clearance after 48-h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion

We previously demonstrated that 48-h isoproterenol (Iso) infusion in rats impaired the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). In this study, we determined whether this impairment persisted over longer time periods by infusing 400 mug.kg(-1).h(-1) Iso by osmotic minipump for 24-144 h (n = 6-7/group). ALC in control rats was 19.0 +/- 2.4 (SD)% of instilled volume absorbed per hour. In Iso-infused rats, ALC was elevated at 24 h (34.9 +/- 2.4%) and decreased at 48 h (15.2 +/- 4.4%) and had recovered to 24 h values at 96 h (37.3 +/- 3.8%) and 144 h (35.2 +/- 3.3%). Plasma Iso concentrations remained elevated at all Iso infusion times. Peripheral lung beta(2)-AR expression exhibited a parallel time course, with a reduction in expression observed at 48 h, followed by an increase to 24 h values at 96 and 144 h. Propranolol prevented the increase in ALC observed at 96 and 144 h, indicating that the recovery in ALC was mediated by a recovery of beta-AR function and beta-AR signaling. ALC at 96 and 144 h could not be further increased by terbutaline, indicating that ALC was maximally stimulated. These data indicate that recovery of beta-AR-stimulated ALC can occur in the continued presence of Iso and is mediated by a recovery of the ability of the distal lung epithelium to respond to beta-AR stimulation.     

Postreceptor defects in alveolar epithelial beta-adrenergic signaling after prolonged isoproterenol infusion

We previously found that prolonged isoproterenol (Iso) infusion in rats impaired the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). Here, we determined if postreceptor defects in beta-AR signaling contribute to this impairment. Iso was infused using subcutaneous miniosmotic pumps (4, 40, or 400 microg. kg-1. h-1) in rats for 48 h. At this time, forskolin-stimulated ALC was measured by mass balance. Forskolin-stimulated ALC [33.4 +/- 2.1%/h (mean +/- SE) in vehicle-infused rats] was reduced by 25 and 38%, respectively, after the 40 and 400 microg. kg-1. h-1 Iso infusions. The ability of forskolin to increase cAMP was reduced by 70% in alveolar type II (ATII) cells isolated from rats infused with 400 microg. kg-1. h-1 Iso. Additionally, the ability of the stable cAMP analog 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer, to increase ALC (48.7 +/- 3.0% in vehicle-infused rats) was reduced by 25 and 51%, respectively, after the 40 and 400 microg. kg-1. h-1 infusions. Finally, the ability of cAMP to increase protein kinase A activity was eliminated in ATII cells isolated from rats infused with Iso at 400 microg. kg-1. h-1. These data demonstrate that prolonged beta-AR agonist exposure can impair alveolar epithelial beta-AR signaling downstream of the beta-AR.     

Prolonged isoproterenol infusion impairs the ability of beta(2)-agonists to increase alveolar liquid clearance

We determined if prolonged isoproterenol (Iso) infusion in rats impaired the ability of the beta(2)-adrenergic agonist terbutaline to increase alveolar liquid clearance (ALC). We infused rats with Iso (at rates of 4, 40, or 400 microg.kg(-1).h(-1)) or vehicle (0.001 N HCl) for 48 h using subcutaneously implanted miniosmotic pumps. After this time, the rats were anesthetized, and ALC was determined (by mass-balance after instillation of Ringer lactate containing albumin into the lungs) under baseline conditions and after terbutaline administration. Baseline and terbutaline-stimulated ALC in vehicle-infused rats averaged, respectively, 19.6 +/- 1.2% (SE) and 44.7 +/- 1.5%/h. The ability of terbutaline to increase ALC was eliminated at 400 microg.kg(-1).h(-1)Iso, inhibited by 26% at 40 microg.kg(-1).h(-1) Iso, and was not affected by 4 microg.kg(-1).h(-1) Iso. beta-adrenergic receptor (betaAR) density of freshly isolated alveolar epithelial type II (ATII) cells from Iso-infused rats was reduced by the 40 and 400 microg.kg(-1).h(-1) infusion rates. These data demonstrate that prolonged exposure to beta-agonists can impair the ability of beta(2)-agonists to stimulate ALC and produce ATII cell betaAR downregulation.     

Effect of epinephrine on alveolar liquid clearance in the rat.

Endogenous epinephrine has been found to increase alveolar liquid clearance (ALC) in several pulmonary edema models. In this study, we infused epinephrine intravenously for 1 h in anesthetized rats to produce plasma epinephrine concentrations commonly observed in this species under stressful conditions and measured ALC by mass balance. Epinephrine increased ALC from 31.5 +/- 3.2 to 48.9 +/- 1.1 (SE)% of the instilled volume (P < 0.05). The increased ALC was prevented by either propranolol or amiloride. To determine whether ALC returns to normal after plasma epinephrine concentration normalizes, we measured ALC 2 h after stopping an initial 1-h epinephrine infusion and found ALC to be at baseline values. Finally, to determine whether desensitization of the liquid clearance response occurs, we evaluated the effects of both repeated 1-h infusions and a continuous 4-h infusion of epinephrine on ALC and found no reduction in ALC under either condition. We conclude that epinephrine increases ALC by stimulating beta-adrenoceptors and sodium transport, that the increase is reversible once plasma epinephrine concentration normalizes, and that desensitization of the ALC response does not appear to occur after 4 h of continuous epinephrine exposure.     

Differential effects of substrate on type I and type II PKA holoenzyme dissociation

It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.     

Discovery of allostery in PKA signaling

Cyclic AMP (cAMP)-dependent protein kinase (PKA) was the second protein kinase to be identified, and the PKA catalytic (C)-subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate many biological functions in eukaryotic cells and are now also a major therapeutic target. The discovery of PKA nearly 50 years ago was quickly followed by the identification of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme. Thus in PKA we see the convergence of two major signaling mechanisms—protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and detailed knowledge of the structure of the isolated regulatory (R)- and catalytic (C)-subunits has been extremely informative. Yet it is the R₂C₂ holoenzyme that predominates in cells, and the allosteric features of PKA signaling can only be fully appreciated by seeing the full-length protein. The symmetry and the quaternary constraints that one R:C heterodimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C heterodimer.     

Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and beta-agonist therapy.

Although keratinocyte growth factor (KGF) protects against experimental acute lung injury, the mechanisms for the protective effect are incompletely understood. Therefore, the time-dependent effects of KGF on alveolar epithelial fluid transport were studied in rats 48-240 h after intratracheal administration of KGF (5 mg/kg). There was a marked proliferative response to KGF, measured both by in vivo bromodeoxyuridine staining and by staining with an antibody to a type II cell antigen. In controls, alveolar liquid clearance (ALC) was 23 +/- 3%/h. After KGF pretreatment, ALC was significantly increased to 30 +/- 2%/h at 48 h, to 39 +/- 2%/h at 72 h, and to 36 +/- 3%/h at 120 h compared with controls (P < 0.05). By 240 h, ALC had returned to near-control levels (26 +/- 2%/h). The increase in ALC was explained primarily by the proliferation of alveolar type II cells, since there was a good correlation between the number of alveolar type II cells and the increase in ALC (r = 0.92, P = 0.02). The fraction of ALC inhibited by amiloride was similar in control rats (33%) as in 72-h KGF-pretreated rats (38%), indicating that there was probably no major change in the apical pathways for Na uptake in the KGF-pretreated rats at this time point. However, more rapid ALC at 120 h, compared with 48 h after KGF treatment, may be explained by greater maturation of alpha-epithelial Na channel, since its expression was greater at 120 than at 48 h, whereas the number of type II cells was the same at these two time points. beta-Adrenergic stimulation with terbutaline 72 h after KGF pretreatment further increased ALC to 50 +/- 7%/h (P < 0.5). In summary, KGF induced a sustained increase over 120 h in the fluid transport capacity of the alveolar epithelium. This impressive upregulation in fluid transport was further enhanced with beta-adrenergic agonist therapy, thus providing evidence that two different treatments can simultaneously increase the fluid transport capacity of the alveolar epithelium.     

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ₂C₂ but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

When part of the heat shock protein DnaJB1 is fused to the catalytic subunit of cAMP-dependent protein kinase (PKA), this fusion protein drives the development of fibrolamellar hepatocellular carcinoma. This study of the asymmetric structure and dynamics of the PKA RIIβ holoenzyme with the oncogenic DnaJB1-PKAc fusion protein reveals disrupted PKA allostery.     

Isoform-specific targeting of PKA to multivesicular bodies

Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase-anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling.     

Identification and characterization of novel PKA holoenzymes in human T lymphocytes

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.     

Control of PKA stability and signalling by the RING ligase praja2

Activation of G-protein-coupled receptors (GPCRs) mobilizes compartmentalized pulses of cyclic AMP. The main cellular effector of cAMP is protein kinase A (PKA), which is assembled as an inactive holoenzyme consisting of two regulatory (R) and two catalytic (PKAc) subunits. cAMP binding to R subunits dissociates the holoenzyme and releases the catalytic moiety, which phosphorylates a wide array of cellular proteins. Reassociation of PKAc and R components terminates the signal. Here we report that the RING ligase praja2 controls the stability of mammalian R subunits. Praja2 forms a stable complex with, and is phosphorylated by, PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteolysis of compartmentalized R subunits, leading to sustained substrate phosphorylation by the activated kinase. Praja2 is required for efficient nuclear cAMP signalling and for PKA-mediated long-term memory. Thus, praja2 regulates the total concentration of R subunits, tuning the strength and duration of PKA signal output in response to cAMP.     

Integrated regulation of PKA by fast and slow neurotransmission in the nucleus accumbens controls plasticity and stress responses

Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. However, the crosstalk between glutamate and dopamine signaling has not been entirely elucidated. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA) via phosphorylation of the PKA regulatory subunit, RIIβ. Using a combination of biochemical, pharmacological, neurophysiological, and behavioral approaches, we find that glutamate-dependent reduction in cyclin-dependent kinase 5 (Cdk5)-dependent RIIβ phosphorylation alters the PKA holoenzyme autoinhibitory state to increase PKA signaling in response to dopamine. Furthermore, we show that disruption of RIIβ phosphorylation by Cdk5 enhances cortico-ventral striatal synaptic plasticity. In addition, we demonstrate that acute and chronic stress in rats inversely modulate RIIβ phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RIIβ regulation by Cdk5 improves behavioral response to stress. We propose this new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.     

Identification and characterization of D-AKAP1 as a major adipocyte PKA and PP1 binding protein

Protein kinase A (PKA) plays an important role in the regulation of lipid metabolism in adipocytes. The activity of PKA is known to be modulated by its specific location in the cell, a process mediated by A-kinase anchoring proteins (AKAPs). In order to examine the subcellular localization of PKA in this tissue we performed a search for AKAP proteins in adipocytes. We purified a 120 kDa protein which can bind both the regulatory subunit of PKA as well as the catalytic subunit of protein phosphatase 1 (PP1). This protein was found to be enriched in the lipid droplet fraction of primary adipocytes and was identified as D-AKAP1. This protein may play an important role in the regulation of PKA in adipocytes.     

PKA: Lessons learned after twenty years

The first protein kinase structure, solved in 1991, revealed the fold that is shared by all members of the eukaryotic protein kinase superfamily and showed how the conserved sequence motifs cluster mostly around the active site. This structure of the PKA catalytic (C) subunit showed also how a single phosphate integrated the entire molecule. Since then the EPKs have become a major drug target, second only to the G-protein coupled receptors. Although PKA provided a mechanistic understanding of catalysis that continues to serve as a prototype for the family, by comparing many active and inactive kinases we subsequently discovered a hydrophobic spine architecture that is a characteristic feature of all active kinases. The ways in which the regulatory spine is dynamically assembled is the defining feature of each protein kinase. Protein kinases have thus evolved to be molecular switches, like the G-proteins, and unlike metabolic enzymes which have evolved to be efficient catalysis. PKA also shows how the dynamic tails surround the core and serve as essential regulatory elements. The phosphorylation sites in PKA, introduced both co- and post-translationally, are very stable. The resulting C-subunit is then packaged as an inhibited holoenzyme with cAMP-binding regulatory (R) subunits so that PKA activity is regulated exclusively by cAMP, not by the dynamic turnover of an activation loop phosphate. We could not understand activation and inhibition without seeing structures of R:C complexes; however, to appreciate the structural uniqueness of each R₂:C₂ holoenzyme required solving structures of tetrameric holoenzymes. It is these tetrameric holoenzymes that are localized to discrete sites in the cell, typically by A Kinase Anchoring Proteins where they create discrete foci for PKA signaling. Understanding these dynamic macromolecular complexes is the challenge that we now face. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

The gamma subunit of Na+, K+-ATPase: role on ATPase activity and regulatory phosphorylation by PKA

In kidney, Na+, K+-ATPase is an oligomer (alphabeta gamma) with equimolar amounts of essential alpha and beta subunits and one small hydrophobic FXYD protein (gamma subunit). This report describes gamma subunit as an activator of pig kidney outer medulla Na+, K+-ATPase in aqueous medium. The effects of gamma subunit on Na+, K+-ATPase were dose-dependent and preincubation-dependent. Changes in alphabeta/gamma stoichiometry did not alter Km1 for ATP, and slightly increased Km2, but Vmax was increased at both catalytic and regulatory sites. Hydroxylamine treatment of enzyme phosphorylated by ATP (E-P), in the presence of additional gamma subunit, revealed that 52% of the E-P accumulation was not via acyl-phosphate formation. The gamma subunit was phosphorylated by endogenous kinases and by commercial catalytic subunit of protein kinase A (PKA). Additionally, we demonstrated that PKA phosphorylation of gamma subunit increased its capacity to stimulate ATP hydrolysis. These results suggest that gamma subunit can act as an intrinsic Na+, K+-ATPase regulator in kidney.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.     

Effects of PKA modulation on the expression of neuropeptide Y in rat amygdaloid structures during ethanol withdrawal

We recently reported that neuropeptide Y (NPY) protein levels and cAMP responsive element binding (CREB) protein phosphorylation are lower in amygdaloid structures during ethanol withdrawal after chronic exposure. Furthermore, we reported that normalization of CREB phosphorylation by infusing protein kinase A (PKA) activator into the central amygdala prevents anxiety-like effects in rats during ethanol withdrawal. Here we investigated whether normalization of CREB phosphorylation by infusing PKA activator (Sp-cAMP) into the central amygdala also normalizes the expression of NPY during ethanol withdrawal. Sprague-Dawley male rats were cannulated targeting the central amygdala and then treated either with Lieber-DeCarli ethanol diet or control diet for 15 days. Subsequently ethanol-fed rats were withdrawn for 0 and 24h. The control-diet fed and ethanol-withdrawn rats were infused twice with PKA activator or inhibitor (Rp-cAMP). The protein and mRNA levels of NPY were determined in amygdaloid structures using gold-immunolabeling and the in situ RT-PCR procedure. It was found that chronic ethanol treatment has no effect on mRNA and protein levels of NPY in the central, medial, or basolateral amygdala. On the other hand, ethanol withdrawal produced significant reductions in mRNA and protein levels of NPY in the central and medial but not in the basolateral amygdala. The reductions in mRNA and protein levels of NPY were normalized in the central amygdala by infusion with PKA activator in ethanol-withdrawn rats. On the other hand, PKA-inhibitor infusion does not have any effect on mRNA and protein levels of NPY in the central amygdala of ethanol-withdrawn rats, but significantly decreased the expression of NPY in the central amygdala of control-diet fed rats. These results suggest that the decreased cellular expression of NPY in the central amygdala may play an important role in the CREB-mediated regulation of anxiety-like behaviors during ethanol withdrawal.     

MAP2 is required for dendrite elongation,PKA anchoring in dendrites,and proper PKA signal transduction

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.     

Evidence for cAMP-dependent protein kinase in the dinoflagellate,Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.