Integrin activation by ìnside-out' signaling is a key process in cell migration and adhesion to extracellular matrix. Recent in-vivo and in-vitro studies have provided convincing evidence that the binding of talin, a cytoskeletal protein, to the integrin [beta] cytoplasmic tails is necessary and sufficient for integrin activation. The significance of its role in integrin signaling leads to the expectation that functions of talin are tightly controlled. Indeed, previous data have shown that cellular distribution of talin and its interaction with integrins is highly regulated; talin resides in the cytosol of platelets under resting conditions but, in response to intracellular signals, translocates to the membrane where it interacts with and activate integrins. However, despite intense interests and vigorous efforts, our understanding of molecular basis of talin regulation remains incomplete. In contrast to FL talin, the N-terminal THD binds to and strongly activates integrins without requiring additional signaling. Thus, we hypothesized that a region in the talin rod domain is involved in suppressing the functions of talin. Sequential C-terminal truncations of the talin rod domain and subsequent mutational analysis revealed that an [alpha]-helical bundle termed Domain E negatively regulates membrane recruitment of talin via its inter- domain interaction with the F3 domain of talin. However, increased membrane association of talin caused by disruption of the Domain E-F3 interaction is insufficient for plasma membrane localization of talin, for the talin- integrin interaction, or for integrin activation in cells or in an in-vitro nanodisc system. NMR analysis revealed an inter-domain interaction between another α- helical bundle in the talin rod called VBS1/2a and the F23 domain of talin, and truncation of VBS1/2a led to increased plasma membrane localization of talin. In addition, truncation analysis identified a short fragment within the linker region between THD and the rod domain that interferes with the talin-integrin interaction. In this study, we have mapped three auto-inhibitory sites within talin and have assigned a specific function to each one of them. These findings provide insights as to how talin is regulated and what may happen to talin during the final steps in integrin activation

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.

The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.