Hilde Abrahamsen

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.

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, β-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 β-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.

Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.

Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1.

Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.

Combined Spatial and Enzymatic Regulation of Csk by cAMP and Protein Kinase A Inhibits T Cell Receptor Signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E2 (PGE2) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE2 reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 ζ-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE2-mediated inhibition of TCR-induced ζ-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.

Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3‐kinase (PI3K‐III) complex and its phosphorylated lipid product phosphatidylinositol 3‐phosphate (PtdIns3P) control the three topologically related membrane‐involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K‐III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1‐associated components are controllable and is regulated by phosphorylation in a context‐dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K‐III complex is controlled by ubiquitination of Beclin 1 or Beclin 1‐associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

ABSTRACT Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-protein kinase A (PKA)-Csk inhibitory pathway that prevents full T-cell activation in the absence of a coreceptor stimulus. Here, we demonstrate that stimulation of CD28 leads to recruitment to lipid rafts of a β-arrestin/phosphodiesterase 4 (PDE4) complex that serves to degrade cAMP locally. Redistribution of the complex from the cytosol depends on Lck and phosphatidylinositol 3-kinase (PI3K) activity. Protein kinase B (PKB) interacts directly with β-arrestin to form part of the supramolecular complex together with sequestered PDE4. Translocation is mediated by the PKB plextrin homology (PH) domain, thus revealing a new role for PKB as an adaptor coupling PI3K and cAMP signaling. Functionally, PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production, leading to recruitment of the supramolecular PKB/β-arrestin/PDE4 complex to the membrane via the PKB PH domain, results in degradation of the TCR-induced cAMP pool located in lipid rafts, thereby allowing full T-cell activation to proceed.

Protein Secretion: Unconventional Exit by Exophagy

Certain secreted proteins bypass the canonical exit pathway from cells. Two studies now shed light on the unconventional secretion route taken by the yeast acyl-coenzyme A-binding protein: this protein is sequestered into autophagic vesicles that are re-routed to the plasma membrane where their content is released to the extracellular space.

Knockdown of C‐terminal Src kinase by siRNA‐mediated RNA interference augments T cell receptor signaling in mature T cells

C‐terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)‐mediated signaling. For the first time, we here report the effects of acuteelimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70–85% knockdown of Csk was achieved within 48 h. No alterations in surface expressionof CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4–5‐fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2–3‐fold higher Lck activity. In Jurkat cells, basal levels of ζ chain phosphorylation were elevated, and spontaneous NFAT‐AP‐1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and ζ chains compared to control. TCR‐induced activation of NFAT‐AP‐1 and TCR/CD28‐stimulated IL‐2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR‐stimulated responses.

Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain

In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser(364) by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.