Phosphodiesterase 3B (PDE3B) antagonizes the anti-angiogenic actions of PKA in human and murine endothelial cells

Recent reports show that protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), acts in a cell autonomous manner to constitutively reduce the angiogenic sprouting capacity of murine and human endothelial cells. Specificity in the cellular actions of individual cAMP-effectors can be achieved when a cyclic nucleotide phosphodiesterase (PDE) enzyme acts locally to control the “pool” of cAMP that activates the cAMP-effector. Here, we examined whether PDEs coordinate the actions of PKA during endothelial cell sprouting. Inhibiting each of the cAMP-hydrolyzing PDEs expressed in human endothelial cells revealed that phosphodiesterase 3 (PDE3) inhibition with cilostamide reduced angiogenic sprouting in vitro, while inhibitors of PDE2 and PDE4 family enzymes had no such effect. Identifying a critical role for PDE3B in the anti-angiogenic effects of cilostamide, silencing this PDE3 variant, but not PDE3A, markedly impaired sprouting. Importantly, using both in vitro and ex vivo models of angiogenesis, we show the hypo-sprouting phenotype induced by PDE3 inhibition or PDE3B silencing was reversed by PKA inhibition. Examination of the individual cellular events required for sprouting revealed that PDE3B and PKA each regulated angiogenic sprouting by controlling the invasive capacity of endothelial cells, more specifically, by regulating podosome rosette biogenesis and matrix degradation. In support of the idea that PDE3B acts to inhibit angiogenic sprouting by limiting PKA-mediated reductions in active cdc42, the effects of PDE3B and/or PKA on angiogenic sprouting were negated in cells with reduced cdc42 expression or activity. Since PDE3B and PKA were co-localized in a perinuclear region in human ECs, could be co-immunoprecipitated from lysates of these cells, and silencing PDE3B activated the perinuclear pool of PKA in these cells, we conclude that PDE3B-mediated hydrolysis of cAMP acts to limit the anti-angiogenic potential of PKA in ECs.     

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium: PHOSPHORYLATION-DEPENDENT INTERACTION OF PDE3A1 WITH SERCA2*

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.     

14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activity

Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking beta-adrenergic signaling and altered human ether-a-go-go related gene (HERG) channel activity. Stress stimulates beta-adrenergic receptors, leading to cAMP elevations that can regulate HERG K+ channels both directly and via phosphorylation by cAMP-dependent protein kinase (PKA). We show that HERG associates with 14-3-3epsilon to potentiate cAMP/PKA effects upon HERG. The binding of 14-3-3 occurs simultaneously at the N- and C-termini of the HERG channel. 14-3-3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14-3-3. The interaction also stabilizes the lifetime of the PKA-phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14-3-3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.     

Cyclic nucleotide phosphodiesterase 3 signaling complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.     

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser(19). This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins gamma, epsilon and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.     

Female infertility in PDE3A-/- mice: Polo-like kinase 1 (Plk1) may be a target of protein kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A-/- mice

Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G₂/M-arrested PDE3A^-/- murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A^-/- oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A^-/- oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A^-/- oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A^-/- mice.Key words: mice oocytes, PDE3A, cAMP, PKA, polo-like kinase 1, centrosome, prophase arrested     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.     

Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding

Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.     

Female infertility in PDE3A-/- mice

Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G2/M-arrested PDE3A^-/- murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A^-/- oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A^-/- oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A^-/- oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A^-/- mice.     

Expression and regulation of cyclic nucleotide phosphodiesterases in human and rat pancreatic islets

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.     

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.     

Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation

Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R(p))-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt(2)cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal.     

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N⁶-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.     

Protein kinase A-mediated 14-3-3 association impedes human Dapper1 to promote dishevelled degradation

Wnt signaling regulates embryo development and tissue homeostasis, and its deregulation leads to an array of diseases, including cancer. Dapper1 has been shown to be a key negative regulator of Wnt signaling. However, its function and regulation remain poorly understood. In this study, we report that 14-3-3β interacts with human Dapper1 (hDpr1). The interaction is dependent on protein kinase A (PKA)-mediated phosphorylation of hDpr1 at Ser-237 and Ser-827. 14-3-3β binding attenuates the ability of hDpr1 to promote Dishevelled (Dvl) degradation, thus enhancing Wnt signaling. We further provide evidence that PKA-mediated Dpr1 phosphorylation may contribute to growth and tumor formation of colon cancer Caco2 cells. Finally, we show that cyclooxygenase-2 expression and PKA activation are positively correlated with Dvl protein levels in colon cancer samples. Together, our findings establish a novel layer of regulation of Wnt signaling by PKA via the 14-3-3-Dpr1-Dvl axis.     

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.     

Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area

The mediation of cAMP effects by specific pools of protein kinase A (PKA) targeted to distinct subcellular domains raises the question of how inactivation of the cAMP signal is achieved locally and whether similar targeting of phosphodiesterases (PDEs) to sites of cAMP/PKA action could be observed. Here, we demonstrate that Sertoli cells of the testis contain an insoluble PDE4D3 isoform, which is shown by immunofluorescence to target to centrosomes. Staining of PDE4D and PKA shows co-localization of PDE4D with PKA-RIIalpha and RIIbeta in the centrosomal region. Co-precipitation of RII subunits and PDE4D3 from cytoskeletal extracts indicates a physical association of the two proteins. Distribution of PDE4D overlaps with that of the centrosomal PKA-anchoring protein, AKAP450, and AKAP450, PDE4D3, and PKA-RIIalpha co-immunoprecipitate. Finally, both PDE4D3 and PKA co-precipitate with a soluble fragment of AKAP450 encompassing amino acids 1710 to 2872 when co-expressed in 293T cells. Thus, a centrosomal complex that includes PDE4D and PKA constitutes a novel signaling unit that may provide accurate spatio-temporal modulation of cAMP signals.     

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.     

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E(2) (PGE(2)) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A(2)/COX-driven autocrine effect whereupon PGE(2) is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

Phosphorylation of the A-kinase-anchoring protein Yotiao contributes to protein kinase A regulation of a heart potassium channel

Regulation of the heart by the sympathetic nervous system, fundamental to the physiological response to stress and exercise, requires coordinated phosphorylation of multiple downstream molecular targets, including the I(Ks) (slowly activating potassium current) channel. Sympathetic nervous system stimulation increases intracellular cAMP for which targeted regulation is directed in large part by distinct scaffold or anchoring proteins. Yotiao is an A-kinase-anchoring protein (AKAP) that recruits the cyclic AMP-dependent protein kinase (protein kinase A (PKA)) and protein phosphatase 1 to the carboxyl terminus of the I(Ks) channel to form a molecular complex and control its phosphorylation state, crucial to the cardiac cellular response to sympathetic nervous system stimulation. Here we report that Yotiao itself is a substrate for PKA phosphorylation, and we identify a Yotiao amino-terminal (N-T) residue (Ser-43) that is PKA-phosphorylated in response to beta-adrenergic receptor stimulation. The replacement of Ser-43 by Ala ablates the PKA phosphorylation of N-T Yotiao and markedly diminishes the functional response of the wild type and pseudo-phosphorylated I(Ks) channel to cAMP but neither prevents the PKA phosphorylation of KCNQ1 nor its binding to Yotiao. These results suggest, for the first time, a critical role for the PKA phosphorylation of an AKAP in the functional regulation of an ion channel protein and postphosphorylation allosteric modulation of the I(Ks) channel by Yotiao.