Shp‐2 Specifically Regulates Several Tyrosine‐Phosphorylated Proteins in Brain‐Derived Neurotrophic Factor Signal

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Abstract
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.