The role of erk and intracellular RHAMM on microtubule stability in transformation

grantor: University of TorontoThe ras/erk pathway is a signaling cascade important in cell growth and motility and, consequently, deregulation of this pathway results in human malignancies including several types of cancer. RHAMMv4 is a key regulator of the ras pathway and enhances erk activation in murine fibroblasts. ' In vitro' studies have previously shown that microtubule (MT)-associated proteins (MAPs) and MT catastrophe factors are potential cytoplasmic targets of erk. Erk-mediated phosphorylation of MAPs has been reported to reduce their ability to stabilize MTs 'in vitro'. Here we show that erk, activated by the ras/MEK pathway, decreases MT stability 'in vivo', as detected by a decrease in acetylated Ã-tubulin levels, a marker for stable MT subsets. Erk decorated MTs in both parental and H-ras transformed murine 10T1/2 fibroblasts and human breast epithelial cells (MCF10a). A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Both PD 98059, a MEK1 inhibitor, and transfection of dominant negative MEK1 cDNA significantly enhanced MT stability in H-ras overexpressing cells, but had no effect in parental cells. In murine fibroblasts, we also observe an 'in vivo' and 'in vitro ' association of RHAMM with MTs in interphase and mitosis, and overexpression of the transforming intracellular isoform, RHAMMv4, causes a decrease in acetylated MTs, compared to parental 10T1/2 cells. RHAMMv4 protein alone does not have a direct destabilizing effect on MTs however the overexpression of RHAMMv4 by transfection studies does enhance erk binding to MTs. Mutations in an erk binding domain of RHAMM abolishes the enhanced MT destabilization and erk binding to MTs observed in RHAMMv4 and H-ras transformed murine fibroblasts. These results provide the first 'in vivo' evidence that active erk promotes MT destabilization in ras-transformed cells and identifies ras as a key upstream regulator of this event. These results further indicate that RHAMM may enhance erk activation and/or association with MTs, potentially bringing this kinase into closer association with MAPs that control MT stability. The resulting MT destabilization likely contributes to the enhanced invasive and proliferative abilities of cells that contain mutant active H-ras and/or overexpress RHAMMv4.Ph.D