Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-T-cell receptor (TCR) stimulation alone is insufficient for activation of T cells, and sustainable T-cell immune responses require a second signal in addition to the TCR-mediated signal. The second signal is typically elicited by ligands B7-1 or B7-2 on antigen-presenting cells engaging the coreceptor CD28 to prevent anergy and apoptosis and enhancing interleukin-2 (IL-2) production and clonal expansion (4). Although CD28 plays a central role in T-cell activation in vivo (5), relatively little is known about the molecular basis for the increased efficacy of T-cell activation upon TCR and CD28 costimulation. Involvement of Lck, Itk, phosphatidylinositol 3-kinase (PI3K), SLP-76, Vav-1, and phospholipase C-␥ (PLC-␥) has, however, been reported (43). CD28-mediated signals are transmitted via a short intracellular stretch in the receptor containing a conserved YMNM motif (32). Phosphorylation of Tyr173 in this motif by Lck and Fyn following CD28 ligation is key to efficient signal transduction (41), generating a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (37, 40). CD28 may also contribute to TCR-dependent PI3K activity without recruiting PI3K directly (18). Whether engagement of CD28 alone can also induce PI3K activity has been a matter of controversy. However, recent reports confirming phosphorylation of the protein kinase B (PKB) substrate glycogen synthase kinase 3 (GSK3) upon CD28 ligation has demonstrated that this is indeed the case (6, 15). In addition, CD28 can recruit growth factor receptor-bound protein 2 (Grb2), and such association of Grb2 occurs via the phosphorylated YMNM motif as well as via the C-terminal PXXP motif (22,35). The PXXP motif also binds and regulates Src family kinases (SFKs) (21, 47), and knock-in mice mutated in this motif were recently reported to have impaired IL-2 secretion (16).Ligation of the TCR induces cyclic AMP (cAMP) production (27). However, the significance of this observation is still not fully understood, as it is well established that cAMP potently inhibits T-cell function and proliferation (2,45,46,50). The spatiotemporal dynamics of the activation-induced cAMP gradient also are not completely appreciated. We have previously shown that cAMP is rapidly produced in lipid rafts following engagement of the TCR in primary T cells (3). This activates a pool of PKA type I targeted to rafts by association with the anchoring protein Ezrin, forming part of a supramolecular complex where Ezrin, EBP50, and PAG provide a scaffold that is able to coordinate PKA phosphorylation and activation of Csk, thereby inhibiting T-cell activation (44, 50). In addition, we have demonstrated that CD3/CD28 costimulation leads to recruitment of type 4 phosphodiesterase (PDE4) isoforms to rafts, resulting in degradation of the TCR-induced cAMP pool (3). Thus, we envisage that TCR-induced cAMP production constitutes a negative feedback loop capable of abrogating T-cell activation ...

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Tyrosine phosphorylation is one of the key regulatory protein modifications in multicellular organisms and tightly controls and coordinates a wide range of cellular responses such as growth, metabolism, tissue repair, migration and apoptosis [1][2][3]. Phosphorylation modulates enzymatic activity as well as creating new binding sites for the recruitment of active molecules into signalling complexes, and assists in building dynamic networks for the transduction of information from the extracellular environment to intracellular signalling pathways. Accurate and specific processing of information is vital to maintaining cellular homeostasis, and errors in signal transduction pathways are linked to a range of diseases such as cancer, autoimmunity and diabetes [4,5].Tyrosine phosphorylation is a reversible modification, regulated by protein tyrosine kinases (PTKs) and phosphatases (PTPs) [6,7]. PTKs of the Src kinase family employ a well-conserved modular arrangement of interaction domains in the regulation of kinase Csk-binding protein ⁄ phosphoprotein associated with glycosphingolipidenriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3-and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp ⁄ PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.Abbreviations Cbp ⁄ PAG, Csk binding protein ⁄ phosphoprotein associated with glycosphingolipids-enriched microdomains; Csk, C-terminal Src kinase; GST, glutathione S-transferase; PPII, polyproline type II; PRD, proline-rich domain; PTK, protein tyrosine kinase; SFK, Src family kinase; SH2, Src homology 2; SH3, Src homology 3; TCR, T-cell receptor.

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