TcR ͞CD3 ligation initiates a signaling cascade involving CD4͞CD8-p56 lck , p59 fyn , and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB͞SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunof luorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.Ligation of CD4͞CD8-p56 lck and the T cell receptor complex (TcR ͞CD3) activates src protein-tyrosine kinases (PTKs) p56 lck and p59 fyn (1), leading to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TcR and CD3 chains (2-4). The phosphorylated ITAM recruits ZAP-70 by means of tandem SH2 domain binding (5, 6). CD4-p56 lck can further up-regulate ZAP-70 catalytic activity by phosphorylation of residue Y-493 (7). The importance of ZAP-70 in T cell signaling has been shown by defects in early signaling events and interleukin 2 (IL-2) transcription in ZAP-70-negative Jurkat cells (8). The Lck-SH2 domain can also bind to ZAP-70, thereby consolidating the CD4͞CD8-p56 lck and TcR ͞CD3 aggregate (9). Consistent with this capability, p56 lck has been found associated with the TcR ͞ CD3 complex (10). p56 lck may also play roles in signaling by other systems by virtue of its ability to phosphorylate other surface receptors such as CD5 and CD28 (11-13).Recent advances have identified downstream targets of the TcR͞CD3 and CD4͞CD8-p56 lck complexes that include hematopoietic proteins VAV, SLP-76, and FYB. VAV possesses several domains, including a guanine nucleotide exchange factor (GEF) domain for the Rho and Rac small GTP-binding proteins (14-16). In turn, VAV binds to another hematopoietic protein, , which possesses a C-terminal SH2 domain and proline-rich motifs (20). ZAP-70 is the key kinase that phosphorylates 22), allowing the SH2 domain to bind to two pYESP motifs (Y-113 and Y-128) within the protein (22). VAV and SLP-76 cooperate to augment TcR ͞CD3 induced IL-2 transcription (17, 18). VAV-SLP-76 complex formation, however, is not essential for TcR-induced IL-2 production in all T cells (22). Instead, SLP-76 requires an intact SLP-76 SH2 domain in its pote...

Protein-tyrosine kinases p56Lck , SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells.

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1’s agonists (Trp-Lys-Tyr-Met-Val-d-Met-CONH2 (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH2 (WRWWWW (WRW4)) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW4 inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW4 to the FPR family, we observed that WRW4 specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid β42 (Aβ42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW4 on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Aβ42 peptide in human neutrophils. Aβ42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW4, which also completely inhibited the internalization of Aβ42 peptide in human macrophages. WRW4 is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.