A palmitoylation switch mechanism regulates Rac1 function and membrane organization.

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.

p38 maintains E-cadherin expression by modulating TAK1-NF-kappa B during epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells is a pathological process that occurs during peritoneal dialysis. EMT leads to peritoneal fibrosis, ultrafiltration failure and eventually to the discontinuation of therapy. Signaling pathways involved in mesothelial EMT are thus of great interest, but are mostly unknown. We used primary mesothelial cells from human omentum to analyze the role of the p38 MAPK signaling pathway in the induction of EMT. The use of specific inhibitors, a dominant-negative p38 mutant and lentiviral silencing of p38α demonstrated that p38 promotes E-cadherin expression both in untreated cells and in cells co-stimulated with the EMT-inducing stimuli transforming growth factor (TGF)-ß1 and interleukin (IL)-1ß. p38 inhibition also led to disorganization and downregulation of cytokeratin filaments and zonula occludens (ZO)-1, whereas expression of vimentin was increased. Analysis of transcription factors that repress E-cadherin expression showed that p38 blockade inhibited expression of Snail1 while increasing expression of Twist. Nuclear translocation and transcriptional activity of p65 NF-κB, an important inducer of EMT, was increased by p38 inhibition. Moreover, p38 inhibition increased the phosphorylation of TGF-ß-activated kinase 1 (TAK1), NF-κB and IκBα. The effect of p38 inhibition on E-cadherin expression was rescued by modulating the TAK1-NF-κB pathway. Our results demonstrate that p38 maintains E-cadherin expression by suppressing TAK1-NF-κB signaling, thus impeding the induction of EMT in human primary mesothelial cells. This represents a novel role of p38 as a brake or 'gatekeeper' of EMT induction by maintaining E-cadherin levels.

Mechanical control of nuclear import by Importin-7 is regulated by its dominant cargo YAP.

Mechanical forces regulate multiple essential pathways in the cell. The nuclear translocation of mechanoresponsive transcriptional regulators is an essential step for mechanotransduction. However, how mechanical forces regulate the nuclear import process is not understood. Here, we identify a highly mechanoresponsive nuclear transport receptor (NTR), Importin-7 (Imp7), that drives the nuclear import of YAP, a key regulator of mechanotransduction pathways. Unexpectedly, YAP governs the mechanoresponse of Imp7 by forming a YAP/Imp7 complex that responds to mechanical cues through the Hippo kinases MST1/2. Furthermore, YAP behaves as a dominant cargo of Imp7, restricting the Imp7 binding and the nuclear translocation of other Imp7 cargoes such as Smad3 and Erk2. Thus, the nuclear import process is an additional regulatory layer indirectly regulated by mechanical cues, which activate a preferential Imp7 cargo, YAP, which competes out other cargoes, resulting in signaling crosstalk.

The absence of caveolin-1 increases proliferation and anchorage- independent growth by a Rac-dependent, Erk-independent mechanism.

Anchorage-independent growth (AIG) of cancer cells requires escape from integrin-mediated signals. A protein frequently downregulated in cancer, caveolin-1 (Cav1), mediates integrin control of several growth-regulatory pathways. We report that loss of Cav1 results in faster exit from quiescence and progress through the cell cycle, proliferation without anchorage to substrate, and absence of cyclin D1 downregulation upon serum deprivation or detachment. Surprisingly, this proliferative advantage is independent of Erk-mitogen-activated protein kinase signaling; instead, cyclin expression and cell cycle progression in the absence of Cav1 are driven by increased membrane order and Rac targeting. AIG was induced in Cav1-expressing cells by forced membrane targeting of Rac1 or by inhibiting Cav1-mediated internalization of plasma membrane ordered domains at which Rac1 accumulates. Restoring Rho activity, which is downregulated after loss of Cav1, antagonizes Rac1 and prevents cyclin D1 accumulation after serum starvation or loss of adhesion. Anchorage independence and increased proliferation in Cav1-deficient tumoral and null cells are thus due to an increased fraction of active Rac1 at membrane ordered domains. These results provide insight into the mechanisms regulating growth of cancer cells, which frequently lose Cav1 function.