| The small GTPase RhoA can cycle between an inactive GDP-bound and an active GTP-bound form. The E3 ubiquitin ligase Smurf1 can bind to nucleotide-free RhoA and regulate the local level of RhoA in cellular protrusions. Smurf1 promotes RhoA ubiquitination, leading to its proteasomal degradation. In kidney podocytes, the actin binding protein synaptopodin induces stress fibers by competitively blocking the Smurf1-mediated ubiquitination of RhoA. Here we show that tropomyosin 2 (Tm2), a ubiquitously expressed actin-binding protein, also regulates RhoA signaling by blocking the ubiquitination of RhoA, thereby establishing the general relevance of this covalent modification. The physiological significance of this mechanism is underscored by the observation that synaptopodin or Smurf1-resistant RhoA(K6,7R) but not wild type RhoA can rescue the loss of stress fibers in Tm2 knockdown fibroblasts and human MDA-MB-231 breast cancer cells lacking Tm2. Functionally, the overexpression of synaptopodin or RhoA(K6,7R) significantly reduces migration of MDA-MB 231 cells in a wound healing assay. Furthermore, transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for Tm2, thereby rescuing germline differentiation and fertility of the mutant. These findings unveil the ubiquitination and proteasomal degradation of RhoA in native and malignant cells as a conserved, regulatory step in the RhoA cycle, in addition to the well-characterized GDP/GTP switch. |
