Identification and characterization of scaffold porteis in JNK cascades

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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.