In resting peripheral T cells, Csk is constitutively present in lipid rafts through an interaction with the Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated leading to dissociation of Csk from lipid rafts. However, tyrosine phosphorylation of PAG/Cbp resumes after 3-5 min, at which time Csk reassociates with the rafts. Cells overexpressing a mutant Csk that lacks the catalytic domain, but displaces endogenous Csk from lipid rafts, have elevated basal levels of TCR--chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses to TCR triggering than controls. We suggest that a transient release from Csk-mediated inhibition by displacement of Csk from lipid rafts is important for normal T cell activation.Activation of the Src family kinases Lck and Fyn after engagement of the T cell antigen receptor is an initiating event in T cell activation and leads to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) 1 within the TCR complex (1). The subsequent recruitment of the tandem SH2 domain containing tyrosine kinase ZAP-70 to phosphorylated ITAMs generates an activated immune receptor signaling complex that is able to initiate downstream events leading to a functional T cell response (2, 3). The control and fine-tuning of the proximal signaling is not only essential for an effective T cell response to antigen, but also for avoiding exaggerated T cell activation and autoimmunity. Thus a balance must be maintained to avoid hypo-as well as hyper-reactivity and immunopathology. The TCR signaling machinery appears to be controlled by setting a threshold for activation to avoid too easy triggering. Suppression of the catalytic activity of Lck and Fyn by phosphorylation of a C-terminal residue (Tyr 505 in Lck, Tyr 528 in Fyn T ) by the C-terminal Src kinase, Csk, appears to be an important means of negative regulation of TCR signaling (4 -6). Complexed with Csk via binding to its SH3 domain is also a protein tyrosine phosphatase, PEP, that dephosphorylates the activating phosphorylation site (Tyr 394 in Lck, Tyr 416 in Fyn T ) (7,8). Recent discoveries indicate that the assembly of TCR signaling complexes occurs in specific membrane subdomains with high cholesterol and glycolipid contents, called glycosphingolipid-enriched microdomains or lipid rafts (9 -11). Key components, including Lck and LAT, are targeted to rafts by virtue of their lipid modifications, whereas other proteins such as the -chain can localize via interaction with raft components only after TCR engagement (11-13). Lipid rafts serve to concentrate and promote specific protein-protein interactions and the tyrosine phosphorylation of signaling intermediates by the Src family kinases during the proximal phases of immunoreceptor signaling via the TCR as well as via the B cell and Fc receptors (14 -16).The ubiquitously expressed, cytosolic Csk ty...

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.

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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.

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.