ASEF2 EXCHANGE ACTIVITY IS STIMULATED BY DYNAMIN2 AND PROMOTES TUMOR CELL POLARIZATION

Abstract

Pancreatic cancer prognosis is poor largely due to the metastatic behavior of tumor cells associated with the disease; therefore, understanding the mechanisms which promote the disruption of cellular cohesion and stimulate migration are of fundamental importance. The Rho family of small GTPases and their associated proteins are central to this subject since they are essential for both the maintenance of cell-to-cell junctions, and activation of proteins involved in actin remodeling and cell migration. Dbl family guanine nucleotide exchange factors are of particular interest since these proteins act as a catalyst to promote GTP-loading and activation of Rho GTPases. In this regard, we have investigated a Cdc42-specific Rho family GEF called Asef2 which we have previously shown to interact with adenomatous polyposis coli (APC) through a discreet motif near the N-terminal region of Asef2. Herein, we describe a novel interaction with Asef2 and Dynamin2, a large GTPase involved in cortical actin remodeling as well as membrane vesicle endocytosis. The interaction between Dynamin2 and Asef2 is mediated through a canonical interaction with the SH3 domain of Asef2 and the proline-rich domain (PRD) of Dynamin2. Moreover, binding of the PRD to the SH3 domain of Asef2 stimulated in vitro exchange on Cdc42, indicating that Dynamin2 releases the auto-inhibition imposed by the SH3 domain. Binding of Asef2 with APC and Dynamin2 was not mutually exclusive, and a ternary complex of the three proteins was identified both in vitro using purified proteins and by co-immunoprecipitation of endogenous cellular protein. Furthermore the proteins were found to co-localize by confocal microscopy, and could be detected at the leading edge of scratch wounded cells. Since APC is known to bind to the microtubule filaments through EB1, and Dynamin2 has been linked to cortical actin through Cortactin, these findings suggest Asef2 acts as a critical linker protein between actin and microtubules during polarized cell migration.