Identification and characterization of scaffold porteis in JNK cascades

MAPK信号转导通路中的支架蛋白将MAPK信号组件组织成功能性MAPK模块，从而实现MAPK通路的高效和特异性激活。我们之前发现了两种新的JNK MAPK结合蛋白，称为JSAP1和JNKBP1，它们可能在MAPK通路中起支架蛋白的作用。在这个研究项目中，我们进一步分析了假定的支架蛋白，得到了以下结果。1.我们已经确定了JSAP1和JNKBP1的家族成员，分别称为JSAP2和JNKBP2。2.我们分析了JSAP1和ASK MAPKKK之间的功能关系。ASK MAPKKK激活JNK和p38 MAPK通路，以响应环境应激，如活性氧。我们的数据表明，JSAP1通过在ASK-JNK信号模块中形成磷酸化依赖的信号复合体来动态参与信号转导。3.我们建立了jsap1缺失的小鼠胚胎干（ES）细胞系。无jsap1胚状体的神经突生长效率明显低于野生型。我们还观察到在jsap1缺失突变体的体外分化过程中分化标志物的表达发生了变化，尤其是神经外胚层标志物。这些结果表明，JSAP1可能是早期胚胎神经发生所必需的。4.我们建立了稳定的PC12h细胞系，表达野生型JSAP1及其缺乏jnk结合域（JBD）的突变体。细胞系的免疫细胞化学研究表明，突变体JSAP1以与野生型JSAP1相似的方式定位在分化的PC12h细胞的生长锥上，这表明JSAP1沿微管的亚细胞定位可能不需要INK信号。

Scaffold proteins in MAP kinase(MAPK) signal transduction pathways organize MAPK signaling components into functional MAPK modules, thereby enabling the efficient and specific activation of MAPK pathways. We previously identified two novel JNK MAPK-binding proteins, termed JSAP1 and JNKBP1, which may function as scaffolding proteins in the MAPK pathways. In this research project, we have further analyzed the putative scaffold proteins, and obtained the following results. 1.We have identified family members of JSAP1 and JNKBP1,termed JSAP2 and JNKBP2,respectively. 2.We have analyzed the functional relationship between JSAP1 and ASK MAPKKK that activates JNK and p38 MAPK pathways in response to environmental stresses such as reactive oxygen species. Our data suggest that JSAP1 dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK-JNK signaling module. 3.We established JSAP1-null mouse embryonic stem(ES) cell lines. Neurite outgrowth from the JSAP1-null embryoid bodies was apparently less efficient than from wild type. We also observed altered expression of differentiation markers, especially markers for neuroectoderm during in vitro differentiation of JSAP1-null mutant. These results suggest that JSAP1 may be required for early embryonic neurogenesis. 4.We established stable PC12h cell lines that expressed wild-type JSAP1 and its mutant lacking the JNK-binding domain(JBD). immunocytochemical studies of the cell lines indicated that the mutant JSAP1 was localized to the growth cones of differentiating PC12h cells in a similar manner to wild-type JSAP, suggesting that the proper subcellular localization of JSAP1 along microtubules probably does not require INK signaling.