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Kjetil Taskén

Publications and source records attributed to Kjetil Taskén.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of A-kinase-anchoring disruptors using a solution-based assay.

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.

A Kinase Anchor Proteins↗
PubMed2006

Enhanced expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental atherosclerosis: possible pathogenic role in plaque destabilization.

OBJECTIVE: Based on their role in T-cell homing into nonlymphoid tissue, we examined the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in coronary artery disease (CAD). METHODS AND RESULTS: We performed studies in patients with stable (n=40) and unstable (n=40) angina and healthy controls (n=20), in vitro studies in T-cells and macrophages, and studies in apolipoprotein-E-deficient (ApoE-/-) mice and human atherosclerotic carotid plaques. We found increased levels of CCL19 and CCL21 within the atherosclerotic lesions of the ApoE-/- mice, in human atherosclerotic carotid plaques, and in plasma of CAD patients. Whereas strong CCR7 expression was seen in T-cells from murine and human atherosclerotic plaques, circulating T-cells from angina patients showed decreased CCR7 expression. CCL19 and CCL21 promoted an inflammatory phenotype in T-cells and macrophages and increased matrix metalloproteinase (MMP) and tissue factor levels in the latter cell type. Although aggressive statin therapy increased CCR7 and decreased CCL19/CCL21 levels in peripheral blood from CAD patients, conventional therapy did not. CONCLUSIONS: The abnormal regulation of CCL19 and CCL21 and their common receptor in atherosclerosis could contribute to disease progression by recruiting T-cells and macrophages to the atherosclerotic lesions and by promoting inflammatory responses in these cells.

Angioplasty, Balloon, Coronary↗
PubMed2006

Molecular basis of AKAP specificity for PKA regulatory subunits.

Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

Adaptor Proteins, Signal Transducing↗
PubMed2006

FOXP3+CD4+CD25+ adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism.

CD4+CD25+ regulatory T (T(R)) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive T(R) (T(R)(adapt)) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. T(R)(adapt) cells produce PGE(2) and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE(2) receptor-specific antagonists. In resting CD4+CD25- T cells, treatment with PGE(2) induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE(2) produced by COX-2-positive T(R)(adapt) cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

Cell Communication↗
PubMed2006

Reduced PDE4 expression and activity contributes to enhanced catecholamine-induced cAMP accumulation in adipocytes from FOXC2 transgenic mice.

Overexpression of forkhead transcription factor FOXC2 in white adipose tissue (WAT) leads to a lean phenotype resistant to diet-induced obesity. This is due, in part, to enhanced catecholamine-induced cAMP-PKA signaling in FOXC2 transgenic mice. Here we show that rolipram treatment of adipocytes from FOXC2 transgenic mice did not increase isoproterenol-induced cAMP accumulation to the same extent as in wild type cells. Accordingly, phosphodiesterase-4 (PDE4) activity was reduced by 75% and PDE4A5 protein expression reduced by 30-50% in FOXC2 transgenic WAT compared to wild type. Thus, reduced PDE4 activity in adipocytes from FOXC2 transgenic mice contributes to amplified beta-AR induced cAMP responses observed in these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

The cyclic AMP-Epac1-Rap1 pathway is dissociated from regulation of effector functions in monocytes but acquires immunoregulatory function in mature macrophages.

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2'-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE(2) inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-alpha production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.

Cell Adhesion↗
PubMed2006

Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor.

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.

Amino Acid Sequence↗
PubMed2006

Immune modulatory effects of cyclooxygenase type 2 inhibitors in HIV patients on combination antiretroviral treatment.

OBJECTIVES: To examine the immune modulating effects of cyclooxygenase type 2 (COX-2) inhibitors (COX-2i) in HIV-infected patients on combination antiretroviral treatment (CART). DESIGN: In-depth substudy from an approved, open, controlled, randomized study comparing the immune modulating effects of CART in combination with COX-2i after 12 weeks. METHODS: Patients (n = 38) on long-term CART with stable viral load (VL) < 50,000 copies/ml and CD4+ T-cell counts > 100/microl were randomized to CART and rofecoxib 25 mg bid (n = 12) or celecoxib 400 mg bid (n = 12), or CART only without placebo (n = 14). Routine clinical chemistry, CD4+ and CD8+ counts and VL were safety parameters. Immunological parameters included C-reactive protein, beta2-microglobulin, Ig isotypes and IgG subclasses as well as several T-lymphocyte subsets. Non-parametric analyses were used throughout. RESULTS: Prestudy experiments showed higher median intracellular expression of COX-2 in CD4+ (P = 0.048) and possibly CD8+ (P = 0.09) T cells from patients on CART compared with uninfected controls. In the clinical study, increased CD4+ T-cell counts were observed only in patients on COX-2i with VL < 50 copies/ml (P = 0.02). Decreased expression of CD38+ on CD8+ T cells and subsets as well as reductions in IgA and IgM (P < 0.03) were most pronounced in patients on COX-2i who had detectable VL (n = 6). COX-2i treatment enhanced the perforin content particularly in the differentiated CD27-/CD8+ T-cell subsets compared with controls (P = 0.05). CONCLUSIONS: COX-2i together with CART improved markers for persistent immune activation, particularly in patients with viraemia, as well as enhanced perforin expression, and thereby strengthened COX-2 as a potential therapeutic target in HIV infection.

ADP-ribosyl Cyclase 1↗
PubMed2005

Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant.

A SNP in the gene PTPN22 is associated with type 1 diabetes, rheumatoid arthritis, lupus, Graves thyroiditis, Addison disease and other autoimmune disorders. T cells from carriers of the predisposing allele produce less interleukin-2 upon TCR stimulation, and the encoded phosphatase has higher catalytic activity and is a more potent negative regulator of T lymphocyte activation. We conclude that the autoimmune-predisposing allele is a gain-of-function mutant.

Alleles↗
PubMed2005

A fast and sensitive method for isolation of detergent-resistant membranes from T cells.

We describe a fast and sensitive method for isolation of detergent-resistant membranes (DRMs) from T cells by sucrose density gradient centrifugation using a smaller accumulated centrifugal force in a tabletop ultracentrifuge. Compared to previous reports, this method, which requires less biological material, is faster and permits quantitative separation of DRMs from other cellular membranes with good resolution. The method, which can be completed in 6 h, yields more than 80% of the total content of DRM-associated adaptor molecules LAT (linker for T cell activation), PAG/Cbp (protein associated with glycosphingolipid-enriched microdomains or Csk-binding protein) and LIME (Lck-interacting membrane protein) in low-density fractions using only 2x10(7) T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

TCR- and CD28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates TCR signaling.

Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or beta-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.

1-Methyl-3-isobutylxanthine↗
PubMed2004

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin.

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.

Actins↗
PubMed2004

The epidermal growth factor-like growth factor amphiregulin is strongly induced by the adenosine 3',5'-monophosphate pathway in various cell types.

We examined the cAMP-mediated regulation of the epidermal growth factor-like growth factor amphiregulin (AR) in T cells and observed a strong cAMP-induced up-regulation of AR mRNA in a time- and concentration-dependent manner independent of T cell activation. This regulation may be mediated in part through activation of a cAMP-responsive element in the AR promoter, because the cAMP-responsive element conferred cAMP responsiveness to a luciferase reporter in Jurkat TAg cells. Similar effects of AR mRNA induction were seen in T cells treated with cAMP-elevating agents such as prostaglandin E(2) and forskolin as well as with the phosphodiesterase inhibitors rolipram and isobutylmethylxanthine. Furthermore, the induction of AR mRNA by cAMP was strongly suppressed by a protein kinase A type I-selective inhibitor, whereas treatment with an exchange protein directly activated by cAMP-specific agonist did not increase AR levels. In addition, an increase in AR gene transcripts by cAMP was seen in MCF-7 mammary carcinoma cells and H295R adrenal cells. Moreover, the potent cAMP-mediated induction of AR mRNA resulted in increased secretion (5-fold) of AR from T cells. Furthermore, supernatants from cAMP-stimulated T cells containing secreted AR induced phosphorylated MAPK in OVCAR-3 carcinoma cells. In conclusion, our data suggest that AR is under strong regulation by the cAMP pathway in various cell types, and that prostaglandin E(2)- and cAMP-induced AR secretion from T cells may be highly relevant in a microenvironment consisting of tumor cells and infiltrated immune cells, because AR by activating the MAPK pathway through a paracrine route may contribute to proliferation of tumor cells and thus add to neoplastic processes.

Adenylyl Cyclases↗
PubMed2004

Haematopoietic protein tyrosine phosphatase (HePTP) phosphorylation by cAMP-dependent protein kinase in T-cells: dynamics and subcellular location.

The HePTP (haematopoietic protein tyrosine phosphatase) is a negative regulator of the ERK2 (extracellular signal-regulated protein kinase 2) and p38 MAP kinases (mitogen-activated protein kinases) in T-cells. This inhibitory function requires a physical association of HePTP through an N-terminal KIM (kinase-interaction motif) with ERK and p38. We previously reported that PKA (cAMP-dependent protein kinase) phosphorylates Ser-23 within the KIM of HePTP, resulting in dissociation of HePTP from ERK2. Here we follow the phosphorylation of this site in intact T-cells. We find that HePTP is phosphorylated at Ser-23 in resting T-cells and that this phosphorylation increases upon treatment of the cells with agents that elevate intracellular cAMP, such as prostaglandin E2. HePTP phosphorylation occurred at discrete regions at the cell surface. Phosphorylation was reduced by inhibitors of PKA and increased by inhibitors of protein phosphatases PP1 and PP2A, but not by inhibitors of calcineurin. In vitro, PP1 efficiently dephosphorylated HePTP at Ser-23, while PP2A was much less efficient. Activation of PP1 by treatment of the cells with ceramide suppressed Ser-23 phosphorylation, as did transfection of the catalytic subunit of PP1. Phosphorylation at Ser-23 is also increased in a transient manner upon T-cell antigen receptor ligation. In contrast, treatment of cells with phorbol ester had no effect on HePTP phosphorylation at Ser-23. We conclude from these results that HePTP is under continuous control by PKA and a serine-specific phosphatase, probably PP1, in T-cells and that this basal phosphorylation at Ser-23 can rapidly change in response to external stimuli. This, in turn, will affect the ability of HePTP to inhibit the ERK and p38 MAP kinases.

Antibodies↗
PubMed2004

Cytokine networks are pre-activated in T cells from HIV-infected patients on HAART and are under the control of cAMP.

OBJECTIVE: Cytokines seem to play a critical role in HIV infection. The cAMP/protein kinase A (PKA) type I pathway is shown to be hyper-activated and contributes to T-cell immune dysfunction in HIV infection. Here, we analysed firstly the levels of cytokine gene expression in unstimulated CD3+T cells from HIV-infected patients on HAART, and secondly the regulation of cytokine and cytokine-related genes by cAMP agonist and antagonist in anti-CD3 activated T cells in order to understand their effects on cytokine networks. METHODS: Cytokine Macro Array and real-time RT-PCR techniques were used to study cytokine gene expression in T cells of HIV-positive patients. RESULTS: Of the cytokine-related genes analysed 45% were expressed at twofold or higher levels in unstimulated T cells from HIV-infected patients as compared with healthy controls, and one-third of these genes were hypo-responsive upon activation as compared with controls. Furthermore, cAMP modulated levels of expression of a number of cytokine-related genes differently in patient and control T cells. CXCR4, CCR5 and amphiregulin were up-regulated by cAMP agonist, whereas other cytokine-related genes including macrophage inflammatory protein 1 beta, tumour necrosis factor-alpha and lymphotoxin-beta were markedly down-regulated by cAMP agonist in T cells from both HIV-infected patients and controls. Moreover, members of the chemokine/chemokine receptor family were over-represented among genes regulated by cAMP agonist/antagonist in patient T cells. CONCLUSIONS: Our data indicate that T cells from HIV-infected patients are in a pre-activated state and that a set of cytokine genes is hypo-responsive to activation and under tonic regulation by cAMP in these T cells.

Adult↗
PubMed2004

Localized effects of cAMP mediated by distinct routes of protein kinase A.

More than 20% of the human genome encodes proteins involved in transmembrane and intracellular signaling pathways. The cAMP-protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells and is involved in regulation of cellular functions in almost all tissues in mammals. Various extracellular signals converge on this signal pathway through ligand binding to G protein-coupled receptors, and the cAMP-PKA pathway is therefore tightly regulated at several levels to maintain specificity in the multitude of signal inputs. Ligand-induced changes in cAMP concentration vary in duration, amplitude, and extension into the cell, and cAMP microdomains are shaped by adenylyl cyclases that form cAMP as well as phosphodiesterases that degrade cAMP. Different PKA isozymes with distinct biochemical properties and cell-specific expression contribute to cell and organ specificity. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein signaling complexes that serve as crossroads for different paths of cell signaling. Targeting of PKA and integration of a wide repertoire of proteins involved in signal transduction into complex signal networks further increase the specificity required for the precise regulation of numerous cellular and physiological processes.

Animals↗
PubMed2004

Effect of activated platelets on expression of cytokines in peripheral blood mononuclear cells - potential role of prostaglandin E2.

Platelets may act as inflammatory cells. To study the effects of soluble and cell-bound platelet factors on the expression of several cytokines and related mediators in leukocytes, peripheral blood mononuclear cells (PBMC) were incubated with platelet-free supernatants from SFLLRN-activated platelet-rich plasma (PRP) or SFLLRN-activated PRP in itself. Our main findings were: (i) the gene expression of several chemokines and some cytokines were markedly increased by both activated PRP and supernatants, as also confirmed at the protein level for IL-6, IL-8 and MIP-1alpha; (ii) the selective protein kinase A type I (PKAI) antagonist Rp-8-Br-cAMP reduced this platelet-induced expression of IL-6, IL-8 and MIP-1alpha in PBMC, suggesting a role of cAMP/PKAI mediated mechanisms in this interaction; (iii) PGE(2) dose-dependently increased the release of IL-6, IL-8 and MIP-1alpha from PBMC mimicking the effect of activated platelets. Furthermore, activated platelets released comparable amounts of PGE(2), suggesting that platelet-derived PGE2 could interact with PBMC in co-cultures; (iv) IL-10 inhibited the platelet-inducing effect on IL-6, IL-8 and MIP-1alpha in PBMC, and notably, the addition PGE2 totally abolished this IL-10 effect suggesting that the suppressive effect of IL-10 on the plateletinduced activation of PBMC might at least partly involve PGE(2) related mechanisms. The present study supports a view of platelets as inflammatory cells, and suggests a potential role of platelet-derived PGE(2) in platelet-induced inflammatory responses.

Adult↗