Analysis of a novel signaling system, the Rap2-MAP4K signaling complex

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We have previously shown that a Ras family small GTP-binding protein Rap2 interacts specifically with MAPKKK kinases (MAP4Ks) that activate the stress-activated MAPK, c-Jun N-terminal kinase (JNK). We believe that Rap2 and MAP4Ks interact with upstream molecules for regulation of Rap2 in response to upstream signals and downstream molecules for transmission of MAP4K signals and that they form a signaling complex, termed Rap2-MAP4K signaling complex. In the present study, we have examined the regulatory mechanisms and cellular functions of this complex, using an epidermal cell line as a model.1. Components and regulatory mechanisms of the signaling complexIn order to identify major components of the signaling complex, we searched for molecules that interact with MAP4Ks. We have identified a protein of approximately 200 kDa (p200) containing the central repeat structure, cystein-rich domain and a C-terminal PDZ-binding motif. This protein appeared to be a mammalian homolog of a Drosophila scaffold protein. We also believe that the complex contains a dimer or oligomer of MAP4Ks.2. Functional analysis of the signaling complexIn fibroblastic cells, Rap2-MAP4K complex regulates cell-substratum adhesion. On the other hand, in epithelial cells, the complex appeared to regulate cell-cell adhesion. We have used a retrovirus system to establish cell lines in that expression of tagged MAP4Ks can be regulated by tetracycline (Tet-Off). Over-expression of MAP4Ks, but not kinase-deficient mutants, negatively regulated cell-cell adhesion.