The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (K D = 3 × 10−8 M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.

CD4 recruitment to T cell receptor (TCR)-peptide-major histocompatibility class II complexes is required for stabilization of low affinity antigen recognition by T lymphocytes. The cytoplasmic portion of CD4 is thought to amplify TCR-initiated signal transduction via its association with the protein tyrosine kinase p56 lck . Here we describe a novel functional determinant in the cytosolic tail of CD4 that inhibits TCR-induced T cell activation. Deletion of two conserved hydrophobic amino acids from the CD4 carboxyl terminus resulted in a pronounced enhancement of CD4-mediated T cell costimulation. This effect was observed in the presence or absence of p56 lck , implying involvement of alternative cytosolic ligands of CD4. A two-hybrid screen with the intracellular portion of CD4 identified a previously unknown 33-kDa protein, ACP33 (acidic cluster protein 33), as a novel intracellular binding partner of CD4. Since interaction with ACP33 is abolished by deletion of the hydrophobic CD4 C-terminal amino acids mediating repression of T cell activation, we propose that ACP33 modulates the stimulatory activity of CD4. Furthermore, we demonstrate that interaction with CD4 is mediated by the noncatalytic ␣/␤ hydrolase fold domain of ACP33. This suggests a previously unrecognized function for ␣/␤ hydrolase fold domains as a peptide binding module mediating protein-protein interactions.The cell surface glycoprotein CD4 is expressed on subsets of thymocytes and mature T lymphocytes and, in humans, on monocytes and macrophages. Early clues to CD4 function came from a strong correlation between CD4 expression and MHC 1 class II restricted T helper cell activity that corresponds to the MHC class I-mediated cytotoxic response of CD8-positive T cells. Subsequent studies using targeted gene disruption in mice and antigen-dependent in vitro T cell activation assays implicated an important role of CD4 in T helper cell development and activation (1, 2).

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.T cell activation is thought to be initiated by the interaction of the T cell antigen receptor (TCR) 1 with two distinct classes of nonreceptor protein-tyrosine kinases (recently reviewed in Refs. 1-3). Biochemical as well as genetic evidence suggests that members of the Src family of nonreceptor protein-tyrosine kinases, represented in T cells by the Fyn and Lck proteins, are involved in this process. Reconstitution experiments of antigen receptor signaling in fibroblasts (4), as well as the biochemical and functional characterization of a Jurkat cell line (JCaM1.6) that lacks Lck kinase activity (5, 6), strengthened the view that Src kinases are responsible for a very important initial step in the antigen receptor signaling cascade, namely the phosphorylation of the intracellular domains of TCR-associated proteins. This includes the homodimer, as well as the CD3 complex composed of the ␥, ␦, and ⑀ chains. With the help of chimeric receptors (7, 8), a distinct motif, now termed ITAM, which is present in all of the above mentioned TCR-associated polypeptides, was found to be sufficient for the induction of T cell activation in various experimental systems (9 -11). The ITAM motif is phosphorylated on tyrosine residues in vivo following antigen receptor stimulation. This phosphorylation event was shown to be responsible for the recruitment of the cytoplasmic ZAP-70 kinase to the phosphorylated receptor via phosphotyrosine-SH2 domain interactions (6,12,13).Surprisingly, significant activation of the ZAP-70 kinase mediated by its binding to ITAMs could not be demonstrated (14), but it was suggested that the recruitment of ZAP-70 into the antigen receptor complex provided a platform for "cross-talk" between the Src and ZAP-70 kinases, which leads to phosphorylation and activation of 16). Co-precipitation analyses demonstrated that ZAP-70 interacts directly with Lck (17-19); furthermore, the protein-tyrosine kinase activity of Lck is required for the phosphorylation of ZAP-70 Tyr 493 in vivo (1...