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

我们以前已经证明，RAS家族的小GTP结合蛋白Rap2与MAPKKK激酶(MAP4Ks)特异性地相互作用，激活应激激活的MAPK，c-Jun氨基末端激酶(JNK)。我们认为，Rap2和MAP4K与上游分子相互作用，调控Rap2信号，下游分子传递MAP4K信号，形成一个信号复合体，称为Rap2-MAP4K信号复合体。在本研究中，我们以一个表皮细胞系为模型，研究了该复合体的调节机制和细胞功能。信号复合体的组成和调控机制为了确定信号复合体的主要成分，我们寻找与MAP4K相互作用的分子。我们已经确定了一个约200 kDa的蛋白质(P200)，它包含中心重复结构、富含半胱氨酸的结构域和C-末端的PDZ结合基序。这种蛋白质似乎是一种果蝇支架蛋白在哺乳动物中的同源物。我们还认为该复合体含有MAP4Ks2的二聚体或寡聚体。成纤维细胞中信号复合体的功能分析，Rap2-MAP4K复合体调节细胞与基质的黏附。另一方面，在上皮细胞中，该复合体似乎调节细胞间的黏附。我们已经使用逆转录病毒系统建立了细胞系，标记的MAP4K的表达可以被四环素(Tet-Off)调控。MAP4Ks的过度表达，而不是激酶缺乏的突变体，负向调节细胞与细胞的黏附。

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.