Anja Ruppelt

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.

Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3ε

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3ε. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3ε to Eps8L1 and Nck SH3 domains. CD3ε bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3ε. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3ε in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3ε to interact with either SH3 or SH2 domain-containing proteins.

Dual Specificity A-kinase Anchoring Proteins (AKAPs) Contain an Additional Binding Region That Enhances Targeting of Protein Kinase A Type I

A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I.

The adaptor protein EBP50 is important for localization of the protein kinase A-Ezrin complex in T-cells and the immunomodulating effect of cAMP.

We recently reported that the dual-specificity AKAP (A-kinaseanchoring protein) Ezrin targets type I PKA (protein kinase A) to the vicinity of the TCR (T-cell receptor) in T-cells and, together with PAG (phosphoprotein associated with glycosphingolipid-enriched membrane microdomains) and EBP50 [ERM (Ezrin/Radixin/Moesin)-binding phosphoprotein 50], forms a scaffold that positions PKA close to its substrate, Csk (C-terminal Src kinase). This complex is important for controlling the activation state of T-cells. Ezrin binds the adaptor protein EBP50, which again contacts PAG. In the present study, we show that Ezrin and EBP50 interact with high affinity (KD=58+/-7 nM). A peptide corresponding to the EB (Ezrin-binding) region in EBP50 (EBP50pep) was used to further characterize the binding kinetics and compete the Ezrin-EBP50 interaction by various methods in vitro. Importantly, loading T-cells with EBP50pep delocalized Ezrin, but not EBP50. Furthermore, disruption of this complex interfered with cAMP modulation of T-cell activation, which is seen as a reversal of cAMP-mediated inhibition of IL-2 (interleukin 2) production, demonstrating an important role of EBP50 in this complex. In summary, both the biochemical and functional data indicate that targeting the Ezrin-EBP interaction could be a novel and potent strategy for immunomodulation.

Negative regulation of T-cell receptor activation by the cAMP-PKA-Csk signalling pathway in T-cell lipid rafts

Spatial organization of signal proteins in specialized cholesterol and glycosphingolipid-enriched microdomains (lipid rafts) provide specificity in lymphocyte signalling. Src kinases associate with lipid rafts on the basis of their dual acylation in the N-terminus and initiate T cell signalling. The immunomodulatory signal enzyme protein kinase A (PKA) is a serine/threonine kinase that controls a number of processes important for immune activation by phosphorylation of substrates that alters protein-protein interactions or changes the enzymatic activity of target proteins in T cells. PKA substrates involved in immune activation include transcription factors, members of the MAP kinase pathway, phospholipases and the Src kinase Csk. The PKA type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal and major target for PKA phosphorylation is the C-terminal Src kinase Csk which initiates a negative signal pathway that fine-tunes the T cell activation process.

A Kinase Anchoring Protein (AKAP) Interaction and Dimerization of the RIα and RIβ Regulatory Subunits of Protein Kinase A In vivo by the Yeast Two Hybrid System

Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.

Mice with Disrupted Type I Protein Kinase A Anchoring in T Cells Resist Retrovirus-Induced Immunodeficiency

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE2- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP–type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5–infected RIAD-transgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP–type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.

Physiological Substrates of PKA and PKG

Publisher Summary This chapter presents the data regarding the total availability of PKA and PKG consensus sites in the human proteome, estimates frequencies of phosphorylation of different motifs, and attempt to give an overview of physiological substrates of both kinases. The cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) belong to the ACG subclass of Ser/Thr-specific protein kinases and generally prefer the phosphate acceptor residue preceded by a row of basic residues. Anchoring proteins (AKAPs, GKAPs) play an important role by locating PKA and PKG in close vicinity to their substrates, and demonstrate how low-affinity substrates may become physiologically relevant. General criteria for identification of physiological substrates of protein kinases are: the target protein should be phosphorylated stoichiometrically and dephosphorylated by phosphatase in vitro at significant kinetic rates; functional properties of the substrate should change in correlation with the degree of phosphorylation; phosphorylation of the substrate should be demonstrated in vivo or in intact cells with accompanying functional changes; the cellular levels of protein kinase should correspond to the extent of phosphorylation of the substrate; and the in situ phosphorylation sequence should be identified. Newly available technologies such as deletion/mutation mapping and mass spectrometry have led to this consensus. Thus the specificity of a substrate is determined not only by the primary sequence, but also by several other factors that affect the degree of phosphorylation of a given target.

Effects of Type I Protein Kinase A Modulation on the T Cell Distal Pole Complex

The distal pole complex (DPC) assembles signalling proteins at the T cell pole opposite the immunological synapse (IS) and is thought to facilitate T cell activation by sequestering negative regulatory molecules away from the T cell receptor‐proximal signalling machinery. Here, we report the translocation of type I protein kinase A (PKA) to the DPC in a fraction of T cells following activation and the localization of type I PKA with known components of the DPC. We propose that sequestration of type I PKA and concomitant loss of cAMP‐mediated negative regulation at the IS may be necessary to allow full T cell activation. Moreover, composition of the DPC appears to be modulated by type I PKA activity, as the antagonist Rp‐8‐Br‐cAMPS inhibited translocation of type I PKA and other DPC proteins.