Loss of ß-adrenergic-stimulated phosphorylation of CaV1.2 channels on Ser1700 leads to heart failure.

L-type Ca2+ currents conducted by voltage-gated calcium channel 1.2 (CaV1.2) initiate excitation-contraction coupling in the heart, and altered expression of CaV1.2 causes heart failure in mice. Here we show unexpectedly that reducing ß-adrenergic regulation of CaV1.2 channels by mutation of a single PKA site, Ser1700, in the proximal C-terminal domain causes reduced contractile function, cardiac hypertrophy, and heart failure without changes in expression, localization, or function of the CaV1.2 protein in the mutant mice (SA mice). These deficits were aggravated with aging. Dual mutation of Ser1700 and a nearby casein-kinase II site (Thr1704) caused accelerated hypertrophy, heart failure, and death in mice with these mutations (STAA mice). Cardiac hypertrophy was increased by voluntary exercise and by persistent ß-adrenergic stimulation. PKA expression was increased, and PKA sites Ser2808 in ryanodine receptor type-2, Ser16 in phospholamban, and Ser23/24 in troponin-I were hyperphosphorylated in SA mice, whereas phosphorylation of substrates for calcium/calmodulin-dependent protein kinase II was unchanged. The Ca2+ pool in the sarcoplasmic reticulum was increased, the activity of calcineurin was elevated, and calcineurin inhibitors improved contractility and ameliorated cardiac hypertrophy. Cardio-specific expression of the SA mutation also caused reduced contractility and hypertrophy. These results suggest engagement of compensatory mechanisms, which initially may enhance the contractility of individual myocytes but eventually contribute to an increased sensitivity to cardiovascular stress and to heart failure in vivo. Our results demonstrate that normal regulation of CaV1.2 channels by phosphorylation of Ser1700 in cardiomyocytes is required for cardiovascular homeostasis and normal physiological regulation in vivo.

Neuronal cAMP/PKA Signaling and Energy Homeostasis.

The brain plays a key role in the regulation of body weight and glucose metabolism. Peripheral signals including hormones, metabolites, and neural afferent signals are received and processed by the brain which in turn elicits proper behavioral and metabolic responses for maintaining energy and glucose homeostasis. The cAMP/protein kinase A (PKA) pathway acts downstream G-protein-coupled receptors (GPCR) to mediate the physiological effects of many hormones and neurotransmitters. Activated PKA phosphorylates various proteins including ion channels, enzymes, and transcription factors and regulates their activity. Recent studies have shown that neuronal cAMP/PKA activity in multiple brain regions are involved in the regulation of feeding, energy expenditure, and glucose homeostasis. In this chapter I summarize recent genetic and pharmacological studies concerning the regulation of body weight and glucose homeostasis by cAMP/PKA signaling in the brain.

Deficiency of the RIIß subunit of PKA affects locomotor activity and energy homeostasis in distinct neuronal populations.

Targeted disruption of RIIß-protein kinase A (PKA) in mice leads to a lean phenotype, increased nocturnal locomotor activity, and activation of brown adipose tissue. Because RIIß is abundantly expressed in both white and brown adipose tissue as well as the brain, the contribution of neuronal vs. peripheral PKA to these phenotypes was investigated. We used a Cre-Lox strategy to reexpress RIIß in a tissue-specific manner in either adipocytes or neurons. Mice with adipocyte-specific RIIß reexpression remained hyperactive and lean, but pan-neuronal RIIß reexpression reversed both phenotypes. Selective RIIß reexpression in all striatal medium spiny neurons with Darpp32-Cre corrected the hyperlocomotor phenotype, but the mice remained lean. Further analysis revealed that RIIß reexpression in D2 dopamine receptor-expressing medium spiny neurons corrected the hyperlocomotor phenotype, which demonstrated that the lean phenotype in RIIß-PKA-deficient mice does not develop because of increased locomotor activity. To identify the neurons responsible for the lean phenotype, we used specific Cre-driver mice to reexpress RIIß in agouti-related peptide (AgRP)-, proopiomelanocortin (POMC)-, single-minded 1 (Sim1)-, or steroidogenic factor 1 (SF1)-expressing neurons in the hypothalamus, but observed no rescue of the lean phenotype. However, when RIIß was reexpressed in multiple regions of the hypothalamus and striatum driven by Rip2-Cre, or specifically in GABAergic neurons driven by Vgat-ires-Cre, both the hyperactive and lean phenotypes were completely corrected. Bilateral injection of adeno-associated virus1 (AAV1)-Cre directly into the hypothalamus caused reexpression of RIIß and partially reversed the lean phenotype. These data demonstrate that RIIß-PKA deficiency in a subset of hypothalamic GABAergic neurons leads to the lean phenotype.

Selective expression of a dominant-negative type Iα PKA regulatory subunit in striatal medium spiny neurons impairs gene expression and leads to reduced feeding and locomotor activity.

Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIß, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIß) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs.

Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity.

MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3'-untranslated regions of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5, and miR-107:CLOCK) were selected from this list, and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets.

Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.

Disruption of the RIIbeta subunit of PKA reverses the obesity syndrome of Agouti lethal yellow mice.

Agouti lethal yellow (A(y)) mice express agouti ectopically because of a genetic rearrangement at the agouti locus. The agouti peptide is a potent antagonist of the melanocortin 4 receptor (MC4R) expressed in neurons, and this leads to hyperphagia, hypoactivity, and increased fat mass. The MC4R signals through Gs and is thought to stimulate the production of cAMP and activation of downstream cAMP effector molecules such as PKA. Disruption of the RIIbeta regulatory subunit gene of PKA results in release of the active catalytic subunit and an increase in basal PKA activity in cells where RIIbeta is highly expressed. Because RIIbeta is expressed in neurons including those in the hypothalamic nuclei where MC4R is prominent we tested the possibility that the RIIbeta knockout might rescue the body weight phenotypes of the A(y) mice. Disruption of the RIIbeta PKA regulatory subunit gene in mice leads to a 50% reduction in white adipose tissue and resistance to diet-induced obesity and hyperglycemia. The RIIbeta mutation rescued the elevated body weight, hyperphagia, and obesity of A(y) mice. Partial rescue of the A(y) phenotypes was even observed on an RIIbeta heterozygote background. These results suggest that the RIIbeta gene mutation alters adiposity and locomotor activity by modifying PKA signaling pathways downstream of the agouti antagonism of MC4R in the hypothalamus.

[Effects of panax quinquefolius saponin of stem and leaf on glucose-lipid metabolism and insulin signal transduction in insulin resistant model adipocytes].

OBJECTIVE: To observe the effects of panax quinquefolius saponin (PQS) of stem and leaf on glucose-lipid metabolism and insulin signal transduction in the insulin resistant model of adipocytes. METHODS: The insulin resistant model of differentiated 3T3-L1 adipocytes was established in vitro with free fatty acid. After induction of insulin resistance, cells were treated with metformin or PQS for 2 days. The glucose consumption in culture fluid was detected by glucose oxidase method; the effects of PQS on the lipolysis induced by tumor necrosis factor (TNF-alpha) was observed using colorimetry; and the phospholation of signal proteins was detected by Western-blot. RESULTS: The amount of glucose consumption (mmol/L) in the model group (5.250 +/- 2. 671) was significantly lower than that in the normal control group (14.133 +/- 1.305, P < 0.01), it increased in the meformin treated group (11.807 +/- 1.358), and the groups treated with high-, middle- and low-dose PQS dose-dependently (10.784 +/- 2.373, 10.217 +/- 1.237 and 9.984 +/- 2.006, respectively), significantly higher than that in the model group (P < 0.01). Upon TNF-alpha treatment, the concentration of free fatty acid (FFA) (nmol/ microg) in culture medium was 2.479 +/- 0.597, predominantly higher than that in the control group (1.320 +/- 0.538, P < 0.01), while it was 1.210 +/- 0.566 in the metformin group, 1.105 +/- 0.631 in high-dose PQS group, 1.108 +/- 0.260 in the middle-dose PQS group, 1.201 +/- 0.593 in the low-dose PQS group, all were lower than that in the TNF-alpha group (P < 0.05 or P < 0.01), and a dose-dependent tendency of PQS's action was seen. The tyrosine phosphorylation of insulin receptor and IRS-1 as well as Ser473 phosphorylation of PKB were lower in the model group than in the control group; they were insignificantly changed in the low-dose PQS group, but did show significant difference in comparing with those in the high-and middle-dose PQS groups or metformin group. CONCLUSION: PQS can accelerate the glucose utilization and depress the lipolysis in adipocytes induced by TNF-alpha, which may be correlated with its promoting insulin signal transduction and improving insulin resistance in adipocytes.

Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy.

In mammals, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is usually elicited by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs). cAMP exerts many of its physiological effects by activating cAMP-dependent protein kinase (PKA), which in turn phosphorylates and regulates the functions of downstream protein targets including ion channels, enzymes, and transcription factors. cAMP/PKA signaling pathway regulates glucose homeostasis at multiple levels including insulin and glucagon secretion, glucose uptake, glycogen synthesis and breakdown, gluconeogenesis, and neural control of glucose homeostasis. This review summarizes recent genetic and pharmacological studies concerning the regulation of glucose homeostasis by cAMP/PKA in pancreas, liver, skeletal muscle, adipose tissues, and brain. We also discuss the strategies for targeting cAMP/PKA pathway for research and potential therapeutic treatment of type 2 diabetes mellitus (T2D).

Hypothalamic PKA regulates leptin sensitivity and adiposity.

Mice lacking the RIIß regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIß knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIß KO mice. During fasting, RIIß-PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIß KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIß KO mice. Our findings suggest that RIIß-PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin.

Mutations in AKAP5 disrupt dendritic signaling complexes and lead to electrophysiological and behavioral phenotypes in mice.

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIalpha or RIIbeta as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and beta-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells.

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase.

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.

Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3.

BACKGROUND: Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. METHODS: Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. RESULTS: Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. CONCLUSIONS: Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.