Kjetil Taskén

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.

FOXC2 Is a Winged Helix Gene that Counteracts Obesity, Hypertriglyceridemia, and Diet-Induced Insulin Resistance

Obesity, hyperlipidemia, and insulin resistance are common forerunners of type 2 diabetes mellitus. We have identified the human winged helix/forkhead transcription factor gene FOXC2 as a key regulator of adipocyte metabolism. Increased FOXC2 expression, in adipocytes, has a pleiotropic effect on gene expression, which leads to a lean and insulin sensitive phenotype. FOXC2 affects adipocyte metabolism by increasing the sensitivity of the beta-adrenergic-cAMP-protein kinase A (PKA) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Increased FOXC2 levels, induced by high fat diet, seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance--a likely consequence hereof would be protection against type 2 diabetes.

Positive and negative regulation of T-cell activation through kinases and phosphatases

The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components.

Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A‐kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21‐22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled‐coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII‐binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA‐buffer extracts of HeLa cells demonstrated co‐precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co‐purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Molecular mechanisms for protein kinase A-mediated modulation of immune function.

Protein kinase A (PKA) is a serine/threonine kinase that regulates a number of cellular processes important for immune activation and control. Modulation of signal transduction by PKA is a complex and diverse process, and differential isozyme expression, holoenzyme composition and subcellular localization contribute specificity to the PKA signalling pathway. In lymphocytes, phosphorylation by PKA has been demonstrated to regulate antigen receptor-induced signalling both by altering protein-protein interactions and by changing the enzymatic activity of target proteins. PKA substrates involved in immune activation include transcription factors, members of the MAP kinase pathway and phospholipases. The ability of PKA type I to regulate activation of signalling components important for formation of the immunological synapse, demonstrates that the cAMP signalling pathway can directly modulate proximal events in lymphocyte activation. Furthermore, the recent discovery that PKA regulates Src kinases through modulation of Csk, supports the notion that PKA is involved in the fine-tuning of immune receptor signalling in lipid rafts.

Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase

The haematopoietic protein tyrosine phosphatase (HePTP) is a negative regulator of the MAP kinases Erk1, Erk2 and p38. HePTP binds to these kinases through a kinase-interaction motif (KIM) in its non-catalytic amino terminus and inactivates them by dephosphorylating the critical phosphorylated tyrosine residue in their activation loop. Here we show that cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine residue 23 in the KIM of HePTP in vitro and in intact cells. This modification reduces binding of MAP kinases to the KIM, an effect that is prevented by mutation of serine 23 to alanine. The PKA-mediated release of MAP kinase from HePTP is sufficient to activate the kinase and to induce transcription from the c-fos promoter. Expression of a HePTP serine-23-to-alanine mutant inhibits MAP-kinase dissociation and activation and induction of transcription from the c-fos promoter. We conclude that HePTP not only controls the activity of MAP kinases, but also mediates crosstalk between the cAMP system and the MAP-kinase cascade.

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 (TR) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive TR (TRadapt) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. TRadapt cells produce PGE2 and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE2 receptor-specific antagonists. In resting CD4+CD25− T cells, treatment with PGE2 induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE2 produced by COX-2-positive TRadapt cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

PI3K p110δ regulates T cell cytokine production during primary and secondary immune responses in mice and humans

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

AKAP complex regulates Ca2+ re‐uptake into heart sarcoplasmic reticulum

The β‐adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca2+‐ATPase (SERCA2), its negative regulator phospholamban (PLN), the A‐kinase anchoring protein AKAP18δ and PKA. We show that AKAP18δ acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca2+ re‐uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca2+ re‐uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

Specificity and spatial dynamics of protein kinase A signaling organized by A-kinase-anchoring proteins

Protein phosphorylation is the most common post-translational modification observed in cell signaling and is controlled by the balance between protein kinase and phosphatase activities. The cAMP-protein kinase A (PKA) pathway is one of the most studied and well-known signal pathways. To maintain a high level of specificity, the cAMP-PKA pathway is tightly regulated in space and time. A-kinase-anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity in the mediation of biological effects controlled by the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphoprotein phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein-signaling complexes.