Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

ZO-1 Regulates Hippo-Independent YAP Activity and Cell Proliferation via a GEF-H1- and TBK1-Regulated Signalling Network.

Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, induced loss of cell density-dependent proliferation control, and promoted apoptosis and necrosis. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on two transcriptional regulators: YAP and ZONAB. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were found to cooperate to activate YAP/TEAD in ZO-1-deficient cells. Thus, ZO-1 controled cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.

Advancements in the Research of GEF-H1: Biological Functions and Tumor Associations.

Guanine nucleotide exchange factor H1 (GEF-H1) is a unique protein modulated by the GDP/GTP exchange. As a regulator of the Rho-GTPase family, GEF-H1 can be activated through a microtubule-depended mechanism and phosphorylation regulation, enabling it to perform various pivotal biological functions across multiple cellular activities. These include the regulation of Rho-GTPase, cytoskeleton formation, cellular barrier, cell cycle, mitosis, cell differentiation, and vesicle trafficking. Recent studies have revealed its crucial effect on the tumor microenvironment (TME) components, promoting tumor initiation and progress. Consequently, an in-depth exploration of GEF-H1's biological roles and association with tumors holds promise for its potential as a valuable molecular target in tumor treatment.

Bone resorption by osteoclasts involves fine tuning of RHOA activity by its microtubule-associated exchange factor GEF-H1.

Bone health is controlled by the balance between bone formation by osteoblasts and degradation by osteoclasts. A disequilibrium in favor of bone resorption leads to osteolytic diseases characterized by decreased bone density. Osteoclastic resorption is dependent on the assembly of an adhesion structure: the actin ring, also called podosome belt or sealing zone, which is composed of a unique patterning of podosomes stabilized by microtubules. A better understanding of the molecular mechanisms regulating the crosstalk between actin cytoskeleton and microtubules network is key to find new treatments to inhibit bone resorption. Evidence points to the importance of the fine tuning of the activity of the small GTPase RHOA for the formation and maintenance of the actin ring, but the underlying mechanism is not known. We report here that actin ring disorganization upon microtubule depolymerization is mediated by the activation of the RHOA-ROCK signaling pathway. We next show the involvement of GEF-H1, one of RHOA guanine exchange factor highly expressed in osteoclasts, which has the particularity of being negatively regulated by sequestration on microtubules. Using a CRISPR/Cas9-mediated GEF-H1 knock-down osteoclast model, we demonstrate that RHOA activation upon microtubule depolymerization is mediated by GEF-H1 release. Interestingly, although lower levels of GEF-H1 did not impact sealing zone formation in the presence of an intact microtubule network, sealing zone was smaller leading to impaired resorption. Altogether, these results suggest that a fine tuning of GEF-H1 through its association with microtubules, and consequently of RHOA activity, is essential for osteoclast sealing zone stability and resorption function.

Lfc subcellular localization and activity is controlled by αv-class integrin.

Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvß3 and α5ß1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvß3 integrin, but not FN-bound α5ß1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvß3 that controls RhoA activity. Mechanistically, FN-engaged αvß3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.

Targeting the RhoA-GEF-H1 pathway of mast cells attenuates experimental airway allergy.

Mast cells are the major effector cells in allergic diseases. RhoA and its downstream pathway is associated with the pathogenesis of airway allergy. The objective of this study is to test a hypothesis that modulating the RhoA-GEF-H1 axis in mast cells can attenuate airway allergy. An airway allergic disorder (AAD) mouse model was employed. Mast cells were isolated from AAD mouse airway tissues to be analyzed by RNA sequencing. We observed that mast cells isolated from the respiratory tract of AAD mice were resistant to apoptosis. Mast cell mediator levels in nasal lavage fluid were correlated with apoptosis resistance in AAD mice. Activation of RhoA in AAD mast cells was related to resistance to apoptosis. Mast cells isolated from the airway tissues in AAD mouse exhibited strong RhoA-GEF-H1 expression. The RhoA-GEF-H1 axis was associated with the lower FasL expression in AAD mast cells. Activation of the RhoA-GEF-H1 axis promoted the production of mediators in mast cells. Inhibition of GEF-H1 facilitated the SIT-induced mast cell apoptosis and enhanced the therapeutic efficacy of AAD. In conclusion, RhoA-GEF-H1 activities are associated with resistance to apoptosis in mast cells isolated from sites of allergic lesions. The state of apoptosis resistance in mast cells is associated with the state of AAD disease. Inhibition of GEF-H1 restores the sensitivity of mast cells to apoptosis inducers, and alleviates experimental AAD in mice.

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Reducing GEF-H1 Expression Inhibits Renal Cyst Formation, Inflammation, and Fibrosis via RhoA Signaling in Nephronophthisis.

Nephronophthisis (NPHP) is the most prevalent monogenic disease leading to end-stage renal failure in childhood. RhoA activation is involved in NPHP pathogenesis. This study explored the role of the RhoA activator guanine nucleotide exchange factor (GEF)-H1 in NPHP pathogenesis. We analyzed the expression and distribution of GEF-H1 in NPHP1 knockout (NPHP1KO) mice using Western blotting and immunofluorescence, followed by GEF-H1 knockdown. Immunofluorescence and renal histology were used to examine the cysts, inflammation, and fibrosis. A RhoA GTPase activation assay and Western blotting were used to detect the expression of downstream GTP-RhoA and p-MLC2, respectively. In NPHP1 knockdown (NPHP1KD) human kidney proximal tubular cells (HK2 cells), we detected the expressions of E-cadherin and α-smooth muscle actin (α-SMA). In vivo, increased expression and redistribution of GEF-H1, and higher levels of GTP-RhoA and p-MLC2 in renal tissue of NPHP1KO mice were observed, together with renal cysts, fibrosis, and inflammation. These changes were alleviated by GEF-H1 knockdown. In vitro, the expression of GEF-H1 and activation of RhoA were also increased, with increased expression of α-SMA and decreased E-cadherin. GEF-H1 knockdown reversed these changes in NPHP1KD HK2 cells. Thus, the GEF-H1/RhoA/MLC2 axis is activated in NPHP1 defects and may play a pivotal role in NPHP pathogenesis.

GEF-H1 Transduces FcεRI Signaling in Mast Cells to Activate RhoA and Focal Adhesion Formation during Exocytosis.

When antigen-stimulated, mast cells release preformed inflammatory mediators stored in cytoplasmic granules. This occurs via a robust exocytosis mechanism termed degranulation. Our previous studies revealed that RhoA and Rac1 are activated during mast cell antigen stimulation and are required for mediator release. Here, we show that the RhoGEF, GEF-H1, acts as a signal transducer of antigen stimulation to activate RhoA and promote mast cell spreading via focal adhesion (FA) formation. Cell spreading, granule movement, and exocytosis were all reduced in antigen-stimulated mast cells when GEF-H1 was depleted by RNA interference. GEF-H1-depleted cells also showed a significant reduction in RhoA activation, resulting in reduced stress fiber formation without altering lamellipodia formation. Ectopic expression of a constitutively active RhoA mutant restored normal morphology in GEF-H1-depleted cells. FA formation during antigen stimulation required GEF-H1, suggesting it is a downstream target of the GEF-H1-RhoA signaling axis. GEF-H1 was activated by phosphorylation in conjunction with antigen stimulation. Syk kinase is linked to the FcεRI signaling pathway and the Syk inhibitor, GS-9973, blocked GEF-H1 activation and also suppressed cell spreading, granule movement, and exocytosis. We concluded that during FcεRI receptor stimulation, GEF-H1 transmits signals to RhoA activation and FA formation to facilitate the exocytosis mechanism.

Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes.

Immune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin-microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance in murine B cells. Our results identify two groups of events, local and global, dominated by actin and microtubules dynamics, respectively. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the lymphocyte-antigen contact site, thereby allowing the formation and maintenance of a unique competent immune synapse.

A molecular clock controls periodically driven cell migration in confined spaces.

Navigation through a dense, physically confining extracellular matrix is common in invasive cell spread and tissue reorganization but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTPase RhoA, which is dependent on the oscillatory changes in the activity and abundance of the RhoA guanine nucleotide exchange factor, GEF-H1, and triggered by a persistent increase in the intracellular Ca2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with a frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

Natriuretic peptide receptor-C releases and activates guanine nucleotide-exchange factor H1 in a ligand-dependent manner.

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.

S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway.

BACKGROUND: Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. RESULTS: We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α. CONCLUSIONS: The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane. Video abstract.

Regulation and functions of the RhoA regulatory guanine nucleotide exchange factor GEF-H1.

Since the discovery by Madaule and Axel in 1985 of the first Ras homologue (Rho) protein in Aplysia and its human orthologue RhoB, membership in the Rho GTPase family has grown to 20 proteins, with representatives in all eukaryotic species. These GTPases are molecular switches that cycle between active (GTP bound) and inactivate (GDP bound) states. The exchange of GDP for GTP on Rho GTPases is facilitated by guanine exchange factors (GEFs). Approximately 80 Rho GEFs have been identified to date, and only a few GEFs associate with microtubules. The guanine nucleotide exchange factor H1, GEF-H1, is a unique GEF that associates with microtubules and is regulated by the polymerization state of microtubule networks. This review summarizes the regulation and functions of GEF-H1 and discusses the roles of GEF-H1 in human diseases.

The LUBAC participates in lysophosphatidic acid-induced NF-κB activation.

The natural bioactive glycerophospholipid lysophosphatidic acid (LPA) binds to its cognate G protein-coupled receptors (GPCRs) on the cell surface to promote the activation of several transcription factors, including NF-κB. LPA-mediated activation of NF-κB relies on the formation of a signalosome that contains the scaffold CARMA3, the adaptor BCL10 and the paracaspase MALT1 (CBM complex). The CBM complex has been extensively studied in lymphocytes, where it links antigen receptors to NF-κB activation via the recruitment of the linear ubiquitin assembly complex (LUBAC), a tripartite complex of HOIP, HOIL1 and SHARPIN. Moreover, MALT1 cleaves the LUBAC subunit HOIL1 to further enhance NF-κB activation. However, the contribution of the LUBAC downstream of GPCRs has not been investigated. By using murine embryonic fibroblasts from mice deficient for HOIP, HOIL1 and SHARPIN, we report that the LUBAC is crucial for the activation of NF-κB in response to LPA. Further echoing the situation in lymphocytes, LPA unbridles the protease activity of MALT1, which cleaves HOIL1 at the Arginine 165. The expression of a MALT1-insensitive version of HOIL1 reveals that this processing is involved in the optimal production of the NF-κB target cytokine interleukin-6. Lastly, we provide evidence that the guanine exchange factor GEF-H1 favors MALT1-mediated cleavage of HOIL1 and NF-κB signaling in this context. Together, our results unveil a critical role for the LUBAC as a positive regulator of NF-κB signaling downstream of LPA receptors.

Microtubule-Mediated NLRP3 Inflammasome Activation Is Independent of Microtubule-Associated Innate Immune Factor GEF-H1 in Murine Macrophages.

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1ß production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1ß maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages.

Go with the flow: GEF-H1 mediated shear stress mechanotransduction in neutrophils.

Neutrophils in circulation experience significant shear forces due to blood flow when they tether to the vascular endothelium. Biochemical and biophysical responses of neutrophils to the physical force of flowing blood modulate their behavior and promote tissue recruitment under pro-inflammatory conditions. Neutrophil mechanotransduction responses occur through mechanisms that are not yet fully understood. In our recent work, we showed that GEF-H1, a RhoA specific guanine nucleotide exchange factor (GEF), is required to maintain neutrophil motility and migration in response to shear stress. GEF-H1 re-localizes to flottilin-rich uropods in neutrophils in response to fluid shear stress and promotes spreading and crawling on activated endothelial cells. GEF-H1 drives cellular contractility through myosin light chain (MLC) phosphorylation downstream of the Rho-ROCK signaling axis. We propose that GEF-H1-dependent cell spreading and crawling in shear stress-dependent neutrophil recruitment from the vasculature are due to the specific localization of Rho-induced contractility in the uropod.

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.

NDR2 kinase contributes to cell invasion and cytokinesis defects induced by the inactivation of RASSF1A tumor-suppressor gene in lung cancer cells.

BACKGROUND: RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown. METHODS: NDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID-/- Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC). RESULTS: We demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC. CONCLUSION: To summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects.