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Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered filamin expression or filamin mutations.

Filamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASBα and that acute degradation of filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2α triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2α-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated.

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. Although many molecular components have been defined, less is known of the formation and regulation of invadopodia. The multidomain protein cortactin, which is involved in the regulation of actin polymerisation, is one such component, but how cortactin is modulated to control the formation of invadopodia has not been elucidated. Here, a new invadopodia synchronization protocol is used to show that the cortactin N-terminal acidic and SH3 domains, involved in Arp2/3 complex and N-WASP binding and activation, respectively, are both required for invadopodia biogenesis. In addition, through a combination of RNA interference and a wide array of cortactin phosphorylation mutants, we were able to show that three convergent regulatory inputs based on the regulation of cortactin phosphorylation by Src-family kinases, Erk1/Erk2 and PAK are necessary for invadopodia formation and extracellular matrix degradation. These findings suggest that cortactin is a scaffold protein bringing together the different components necessary for the formation of the invadopodia, and that a fine balance between different phosphorylation events induces subtle changes in structure to calibrate cortactin function.

The integral membrane type 1 matrix metalloprotease (MT1-MMP) is a pivotal protease in a number of physiological and pathological processes and confers both non-tumorigenic and tumorigenic cell lines with a specific growth advantage in a three-dimensional matrix. Here we show that, in a melanoma cell line, the majority (80%) of MT1-MMP is sorted to detergent-resistant membrane fractions; however, it is only the detergent-soluble fraction (20%) of MT1-MMP that undergoes intracellular processing to the mature form. Also, this processed MT1-MMP is the sole form responsible for ECM degradation in vitro. Finally, furin-dependent processing of MT1-MMP is shown to occur intracellularly after exit from the Golgi apparatus and prior to its arrival at the plasma membrane. It is thus proposed that the association of MT1-MMP with different membrane subdomains might be crucial in the control of its different activities: for instance in cell migration and invasion and other less defined ones such as MT1-MMP-dependent signaling pathways.