Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival of various types of neurons in the central nervous system. BDNF binds to and activates the tyrosine kinase receptor, TrkB, initiating intracellular signaling and exerting its effects. Phosphatidylinositol 3-kinase (PI3-K), which has been implicated in promotion of neuronal survival by neurotrophic factors, is a component in the signaling pathway of BDNF. We examined how BDNF activates PI3-K in cultured cerebral cortical neurons. We found that insulin receptor substrate (IRS)-1 and -2 are involved in the BDNF signaling pathway that activates PI3-K. IRS-1 and -2 were tyrosine-phosphorylated and bound to PI3-K in response to BDNF. This BDNFstimulated signaling via IRS-1 and -2 was inhibited by K-252a, an inhibitor of Trk tyrosine kinase. In addition, signaling via IRS-1 and -2 was markedly sustained as well as the BDNF-induced tyrosine phosphorylation of TrkB. On the other hand, we observed no association of PI3-K with TrkB in response to BDNF. These results indicate that the activation of TrkB by BDNF induces the activation of PI3-K via IRS-1 and -2 rather than by a direct interaction of TrkB with PI3-K in cultured cortical neurons.

BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in gt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.Protein tyrosine phosphorylation plays an important role in signal transduction and regulates a wide range of cellular processes. Protein-tyrosine kinases and protein-tyrosine phosphatases are highly expressed in the central nervous system, consistent with the importance of tyrosine phosphorylation in neural function (1).BIT 1 (a brain immunoglobulin-like molecule with tyrosinebased activation motifs) is a novel immune antigen receptorlike molecule of the brain.2 This molecule is composed of an antigen receptor-like extracellular domain, a transmembrane domain, and a cytoplasmic region containing two variants of TAM (tyrosine-based activation motif) that was recently designated ITAM (immunoreceptor TAM). This cytoplasmic motif contains two tyrosine phosphorylation sites. TAM was originally described in the immune system where it plays a crucial role in the activation responses of B and T cells (2-5). BIT is one of major substrates of protein-tyrosine kinase(s) in crude brain suspensions 2 and is widely distributed in the brain in synapse-rich regions and in some nerve fibers.3 These findings suggest that the tyrosine phosphorylation of TAMs in BIT may be involved in neural signal transduction. Recent studies in the immune system have demonstrated that the oligomerization of TAMs allows the phosphorylation of two tyrosine residues found in this motif and these phosphotyrosine residues act as a bidentate docking site for the paired Src homology 2 (SH2) domains present in the cytoplasmic tyrosine kinases, Syk and ZAP-70, believed to be indispensable for initiation of the signaling cascade (6 -8). From these investigations, we predicted that TAMs of BIT may recruit tyrosine kinases containing paired SH2 domains to the ...

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival and regulates plasticity of various types of neurons. BDNF binds to TrkB, a receptor tyrosine kinase, which results in the activation of a variety of signaling molecules to exert the various functions of BDNF. Shp-2, a Src homology 2 domain-containing cytoplasmic tyrosine phosphatase, is involved in neurotrophin signaling in PC12 cells and cultured cerebral cortical neurons. To examine the roles of Shp-2 in BDNF signaling in cultured rat cerebral cortical neurons, the wild-type and phosphatase-inactive mutant (C/S mutant) forms of Shp-2 were ectopically expressed in cultured neurons using recombinant adenovirus vectors. We found that several proteins tyrosinephosphorylated in response to BDNF showed enhanced levels of tyrosine phosphorylation in cultured neurons infected with C/S mutant adenovirus in comparison with those infected with the wild-type Shp-2 adenovirus. In addition, in immunoprecipitates with anti-Shp-2 antibody, we also observed at least four proteins that displayed enhanced phosphorylation in response to BDNF in cultured neurons infected with the C/S mutant adenovirus. We found that the Shp-2-binding protein, brain immunoglobulin-like molecule with tyrosine-based activation motifs (BIT), was strongly tyrosine-phosphorylated in response to BDNF in cultured neurons expressing the C/S mutant of Shp-2. In contrast, the level of BDNF-induced phosphorylation of mitogen-activated protein kinase and coprecipitated proteins with anti-Trk and Grb2 antibodies did not show any difference between neurons infected with these two types of Shp-2. Furthermore, the survival effect of BDNF was enhanced by the wild type of Shp-2, although it was not influenced by the C/S mutant of Shp-2. These results indicated that in cultured cerebral cortical neurons Shp-2 is specifically involved in the regulation of several tyrosine-phosphorylated proteins, including BIT, in the BDNF signaling pathway. In addition, the phosphatase Shp-2 may not influence the level of BDNF-induced activation of mitogen-activated protein kinase in cultured cortical neurons. Further, Shp-2 may have potential to positively regulate BDNF-promoting neuronal survival. Key Words: Neurotrophin-Adenovirus vector-Tyrosine phosphatase-TrkB.

Brain-derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1) is a substrate of Shp-2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS-1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS-1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS-1 were replaced with phenylalanine. Overexpression of wild-type BIT/SHPS-1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/ SHPS-1 itself and an association of Shp-2 with BIT/ SHPS-1 even without addition of BDNF. We found that BDNF-promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS-1 does not depend on its tyrosine phosphorylation. BDNF-induced activation of mitogen-activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF-induced activation of Akt was enhanced in neurons expressing wild-type or 4F mutant BIT/SHPS-1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, blocked the enhancement of BDNF-promoted neuronal survival in both neurons expressing wild-type and 4F mutant BIT/SHPS-1. These results indicate that BIT/SHPS-1 contributes to BDNFpromoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3-kinase-Akt pathway. Our results suggest that a novel action of BIT/SHPS-1 does not occur through tyrosine phosphorylation of BIT/SHPS-1 in cultured cerebral cortical neurons.

A monoclonal antibody, 1D4, recognizing a novel brain-specific protein was obtained. The 1D4 antigen is regarded to be a glycoprotein because it was adsorbed on the Con A-Sepharose column used for its purification. The antiserum (polyclonal antibodies) against the 1D4 antigen was raised in a rabbit and shown to react with just the same molecules as the 1D4 monoclonal antibody did. It was used to detect the antigen in crude tissue homogenates. The molecular mass of the 1D4 antigen was estimated to be 89 kDa by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the brain homogenate. The 1D4 antigen had multiple isoelectric points, the pattern of the bands detected on isoelectric focusing gel being quite similar to that of Type B nucleoside diphosphatase of the brain. However, they are distinct, since Type B nucleoside diphosphatase was not adsorbed by anti-1D4 antigen IgG-Sepharose 4B. The 1D4 antigen could not be detected in any of the peripheral organs or tissues tested. The 1D4 antigen was rich in the cerebrum, diencephalon, and cerebellum in the brain, and its content decreased with the distance of the region from the cerebrum. The amounts of the 1D4 antigen in the cerebrum and cerebellum increased with the respective developmental maturation. These findings suggest that the 1D4 antigen contributes to some brain-specific functions of the mature brain.