The Pennsylvania State University

Epithelial cells form largely non-motile structures in most circumstances. However, wound healing and developmental processes require epithelial cell motility in normal physiological conditions. In other instances, cell migration plays a role in pathobiological conditions such as metastases of cancers generating from epithelial tissues. Understanding the triggers and the mechanisms for migration is therefore crucial in attempting to prevent metastasis in cancer or promote it in wound healing. The hepatocyte growth factor, HGF, is a potent inducer of motility for epithelial cells. It activates its receptor the c-Met tyrosine kinase and induces downstream cascades that promote movement. In this study we show that HGF signaling depends on a signal transduction cascade that involves multiple small GTPases downstream of c-Met. We identify two scaffolding proteins, IPCEF/CNK3 and GRASP/Tamalin necessary for assembling a multiprotein complex that brings together Arf6 and Rac1 and their respective GEFs, ARNO and Dock180 to a cellular location where they promote cytoskeletal changes and induce motility. We first identify the role of IPCEF1/CNK3 in this cascade. IPCEF and CNK3 were first thought to be two separate proteins. We found that they are two halves of the same protein. This one protein is homologous to CNK in Drosophila melanogaster. CNK3 not only interacts with ARNO and enhances its activity, but as we demonstrate, has the capacity of anchoring ARNO in a location where it can activate Arf6 downstream of HGF. We subsequently studied the function of GRASP/Tamalin in this multiprotein complex mediating Arf-to-Rac signaling. We found that GRASP acts as the molecular bridge between ARNO and Dock180, facilitating the cascade that promotes cell migration downstream of HGF. Knockdown of GRASP was shown to have detrimental effects on HGF-induced cell migration in iv general. In addition preliminary data suggest that the absence of GRASP negatively affects the activation of Rac1. These data allow us to describe the importance of protein-protein interactions in the context of cell migration. Despite the absence of catalytic domains on scaffolding proteins, they play an important role in locating the signal and connecting receptor tyrosine kinase signaling to small GTPases crosstalk. The activation of small GTPases does not give a complete picture and definitely does not answer all the questions as to where, when and why cells elect to migrate or not. Scaffolding proteins fill in certain gaps in understanding the fine tuning required for proper protein assembly at the right time and location inside the cell.