Abstract:The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuronastrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the ␤-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function. Key Words: Vasoactive intestinal peptide-Apolipoprotein E-Learning and memory-Neuronal survival-Molecular cloning-mRNA.

T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.

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T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion-and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.T cell activation triggers multiple molecular events, including the activation of protein tyrosine kinases (PTKs), formation of multiprotein signaling complexes, and activation of enzymes and transcription factors (25,37,47). Cytoskeletal actin reorganization is also dependent on these events initiated at the T cell-antigen-presenting cell (APC) interface, the immunological synapse (IS). Interference with actin dynamics results in an impaired immune response and can induce T cell anergy (40).We and others (2,4,8,11,12,16) have demonstrated complex molecular events linking T cell antigen receptor (TCR) activation to actin rearrangement. One major pathway, mediated by the activation of multiple PTKs, leads to phosphorylation of the adapter molecules linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). Phosphorylation of SLP76 leads to recruitment of the Nck adapter molecule, which is associated with key regulators of the actin cytoskeleton Wiskott-Aldrich syndrome protein (WASp) and WAVE2.The molecular structure of SLP76 consists of an N-terminal sterile-alpha motif (SAM) (41), an acidic domain containing tyrosine residues subject to phosphorylation, a central prolinerich region, and a C-terminal SH2 domain. Phosphorylation of the tyrosines allows the interaction of SLP76 with the adapter Nck, the Rho-family GEF, VAV, and Itk, all via their SH2 domains (5,8,48,49,51). The interactions of SLP76, Nck, and VAV are essential for the activation of WASp and its recruitment to the IS (51). TCR engagement also induces the association of the SLP76 SH2 domain with the adhesion-and degranulation-promoting adapter protein (ADAP) and to the serine-threonine kinase hematopoietic progenitor kinase 1 (HPK-1) (38). In addition to SLP76, ADAP is capable of binding other proteins, and it is recruited to the IS (26, 35). The role of ADAP in integrin function has been explored (10,23,36,46); however, it...