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.

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.

We report here the cloning of a chicken cDNA (402 aa) showing high sequence similarity to the previously cloned rat and human P2X 5 receptors (67 and 69%, respectively). The chicken P2X 5 subunit is encoded by a gene composed of 12 translated exons, which shows conserved genomic structure with mammalian P2X genes. In HEK-293 cells heterologously expressing chicken P2X 5 receptors, ATP activates a current that desensitizes in a way that is dependent on the presence of extracellular divalent cations. ATP and 2-methylthio ATP are equipotent agonists (EC 50 , 2 mM) and suramin and pyridoxal 5-phosphate-6-azophenyl-2 H ,4 H -disulfonic acid are potent antagonists. Additionally, reversal potential measurements indicate that chicken P2X 5 is permeable not only to cations but also to chloride (P Cs1 /P Cl-, 1.9), as has been described for native P2X receptor mediated responses in embryonic chicken skeletal muscle. mRNA distribution of chicken P2X 5 was determined by in situ hybridization analysis in both whole embryos and on tissue slices of heart and skeletal muscle. Our results suggest that chicken P2X 5 receptors are expressed in developing muscle and might play a role in early muscle differentiation.

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.

The second messenger cAMP is frequently utilized in mammalian cells to regulate a variety of physiological processes. Cyclic AMP is generated at the plasma membrane in response to the occupancy of G-protein-coupled receptors. This ultimately leads to the stimulation of adenylyl cyclases, the enzymes that produce cAMP. The newly synthesized cAMP diffuses into the cell where it is available to activate a variety of effector proteins. These include protein kinase A (PKA) 4 (reviewed in Ref. 1), cAMP-regulated ion channels (2), and Epac guanine nucleotide exchange factors (3). Activation of the PKA holoenzyme occurs upon binding of cAMP to the regulatory (R) subunits. This promotes dissociation of the active catalytic (C) subunits from the tetrameric complex and results in the phosphorylation of substrates in the vicinity of the active kinase (4, 5). PKA holoenzymes are classified as either type I or type II on the basis of their R subunit composition (RI or RII) (6). Four genes encode R subunits (RI␣, RI␤, RII␣, and RII␤). These proteins have distinct physical properties and affinities for cAMP (1). Because PKA is a broad specificity serine/threonine protein kinase that regulates a wide range of cellular processes, additional mechanisms have evolved to influence the selectivity of PKA action (7). Specificity in PKA action is maintained in part by interaction with protein kinase A anchoring proteins (AKAPs). This family of structurally diverse but functionally related scaffolding proteins targets PKA and other signaling proteins toward distinct substrates. These protein-protein targeting interactions contribute to spatial and temporal regulation of second messenger signaling events (reviewed in Refs. 7,8).The AKAP family now includes more than 50 members when including splice variants (7,8). Although most of the AKAPs were initially identified on the basis of their ability to bind PKA type II inside cells, it is now recognized that several of these anchoring proteins such as D-AKAP1, D-AKAP2, AKAP220, Ezrin, Merlin, and PAP7 have a dual specificity as they also bind PKA type I (9 -14). Other AKAPs are reported to selectively bind RI such as AKAP CE , myosin, and ␣4 integrins (15-17). However, only two of these dual specificity proteins, the mitochondrial protein PAP7 and Ezrin (12,18), have been shown to preferentially interact with PKA type I in situ (15)(16)(17).Conventional AKAPs contain a conserved amphipathic helix of 14 -18 residues that forms the PKA-anchoring domain (19 -21). This region inserts into a hydrophobic groove formed by the R dimer (22, 23). The RII subunits dimerize at the N terminus in an antiparallel fashion forming an X-type four-helix bundle that is necessary for AKAP binding. RI contains a structur-