Regulation of Cadherins by MLK3

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ABSTRACT Mixed lineage kinase (MLK) 3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that regulates multiple MAPK signaling pathways in mammalian cells. In addition, MLK3 has been demonstrated to regulate proliferation and invasion of different types of human epithelial cells (normal and cancer) including ovarian and breast. In the current study, we identified a novel function for MLK3 in cell-cell adhesion of ovarian epithelial TOV112D tumor cells. MLK3 siRNA knockdown disrupted cell-cell adhesion in TOV112D spheroids, reduced the cell junction protein, E-cadherin, and dramatically increased the level of Slug, a transcriptional repressor of E-cadherin. Furthermore, MLK3 was co-immunoprecipitated with E-cadherin in TOV112D cell lysates, which indicates that MLK3 may be associated with adherens junction protein complexes. Additionally, MLK3 overexpression induced the cleavage of E-cadherin resulting in an 80 kDa soluble form of E-cadherin, which has been found to activate growth factor receptor signaling. In TOV112D cells, MLK3 overexpression also strongly induced the expression of N-cadherin, a cell junction protein that is normally expressed in mesenchymal cells. Our central hypothesis is that MLK3 regulates E-cadherin and N-cadherin through multiple mechanisms, and this regulation is essential for proper cell adhesion and migration in ovarian epithelial cells. The proposed experiments are designed to define the mechanism(s) by which MLK3 regulates E- and N- cadherin, and the impact of this regulation on ovarian cell spheroid adhesion and migration. Three specific aims are proposed to test the hypothesis. In Aim 1, we will investigate MLK3 functions in E-cadherin multi- protein complexes and ovarian spheroid cell-cell adhesion. In aim 2, we will examine MLK3-dependent regulation of Slug and E-cadherin expression. In Aim 3, we will evaluate MLK3-dependent regulation of E- Cadherin cleavage, N-Cadherin expression and ovarian spheroid structure. This research will reveal novel insight into the mechanisms by which MLK3 functions to regulate E- and N-cadherin and modulate cell-cell adhesion and migration, processes which are essential for development, tissue morphogenesis, migration and cancer cell invasion.