SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.     

Novel mechanism of the co-regulation of nuclear transport of SmgGDS and Rac1

The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS. Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS.RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.     

Di-Ras2 Protein Forms a Complex with SmgGDS Protein in Brain Cytosol in Order to Be in a Low Affinity State for Guanine Nucleotides

The Ras family of small GTPases function in a wide variety of biological processes as “molecular switches” by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

Identification and characterization of the unique guanine nucleotide exchange factor, SmgGDS, in vascular smooth muscle cells

The guanine nucleotide exchange factor (GEF), SmgGDS, promotes nucleotide exchange by several GTPases in both the Ras and Rho families, especially by RhoA. Because RhoA plays an important role in regulating the contraction of vascular smooth muscle cells (VSMC), we examined the expression and function of SmgGDS in VSMC. SmgGDS is expressed in primary rat aortic smooth muscle (ASM) cells, primary bovine coronary artery smooth muscle (BCASM) cells, and the immortalized A7r5 line of rat ASM cells. Down regulation of SmgGDS expression by siRNA transfection resulted in a decrease of RhoA-GTP levels, enhanced cell spreading, and loss of the characteristic elongated morphology of VSMC. A similar morphology was also observed following treatment with the Rho-kinase inhibitor, Y27632. In contrast, cells with reduced RhoA expression exhibit an elongated shape. Subsequent immunofluorescent staining revealed a disruption of the myosin filament organization in the cells with reduced SmgGDS expression. Further studies analyzed the effect of SmgGDS siRNA transfection on the contraction of A7r5 cells and BCASM cells, which is also a Rho-regulated pathway. Transfection of SmgGDS siRNA or RhoA siRNA resulted in an impaired ability of the A7r5 and BCASM cells to undergo contraction in a collagen gel matrix. However, phosphorylation of the myosin-binding subunit of myosin phosphatase (MYPT1) or the light chain of myosin II (MLC) was not altered by downregulating expression of either SmgGDS or RhoA GTPase. Taken together these results identify SmgGDS as a novel regulator of myosin organization and contraction in VSMC.     

Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 X 10(-5) s(-1), considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.     

The activation of Rac1 by M3 muscarinic acetylcholine receptors involves the translocation of Rac1 and IQGAP1 to cell junctions and changes in the composition of protein complexes containing Rac1, IQGAP1, and actin

The abilities of the M(3) muscarinic acetylcholine receptor (mAChR) and Rac1 to regulate similar cellular responses, including cadherin-mediated adhesion, prompted us to investigate Rac1 regulation by M(3) mAChR. We characterized changes in Rac1 induced by stimulating transfected M(3) mAChR in Chinese hamster ovary cells stably expressing hemagglutinin (HA)-tagged wild-type or mutant Rac1. mAChR activation converts endogenous Rac1 to the GTP-bound form in cells expressing HA-Rac1 but not in cells expressing dominant negative HA-Rac1(Asn-17) or constitutively active HA-Rac1(Val-12). The competitive binding of endogenous IQGAP1 by HA-Rac1(Val-12) may diminish the mAChR-mediated activation of endogenous Rac1. HA-Rac1 and HA-Rac1(Val-12), but not HA-Rac1(Asn-17), accumulate with IQGAP1 at cell junctions during mAChR-induced cell-cell compaction. Co-localization studies suggest that Rac1 can accumulate at junctions without IQGAP1, but IQGAP1 cannot accumulate at junctions without Rac1. mAChR activation also induces GTP-independent changes in Rac1 because mAChR activation redistributes HA-Rac1(Asn-17), which does not bind GTP. Actin associates with complexes containing HA-Rac1 or HA-Rac1(Val-12) after prolonged mAChR activation. We also demonstrate that Rac1 participates in mAChR-induced cell-cell compaction and c-Jun phosphorylation. These results indicate that M(3) mAChR activation converts Rac1 to the GTP-bound form, alters interactions between Rac1, IQGAP1, and actin, and causes the junctional accumulation of Rac1 and IQGAP1.     

Redox regulation of RhoA

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).     

The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals

Background

Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.

Principal Findings

Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.

Conclusions/Significance

These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.     

Mapping Interactions between p27 and RhoA that Stimulate Cell Migration

p27 mediates cell cycle arrest by binding to and inhibiting cyclin-dependent kinase/cyclin complexes, but p27 can also contribute to pro-oncogenic signaling upon mislocalization to the cytoplasm. Cytoplasmic p27 stimulates cell migration by associating with RhoA and interfering with the exchange of GDP from RhoA stimulated by guanine nucleotide exchange factors. We used biophysical methods to show that the N-terminus of p27 directly interacts with RhoA in vitro. The affinity of p27 for RhoA is low, with an equilibrium dissociation constant of hundreds of micromolar; however, at high concentrations, p27 interfered with guanine nucleotide exchange factor-mediated nucleotide exchange from RhoA. We also show that promotion of cell migration in scratch wound cell healing assays requires full-length p27 despite the C-terminus being dispensable for the direct interaction between p27 and RhoA in vitro. These results suggest that there may be an unidentified factor(s) that associates with the C-terminus of p27 to enhance its interactions with RhoA and promote cell migration.     

Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.     

EspM2 is a RhoA guanine nucleotide exchange factor

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide‐free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H‐Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.     

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP. G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP. G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP. RhoA to GTP. RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP. RhoA.     

Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras.

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.     

PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 micro M forskolin plus 1 micro M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of (32)P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of approximately 23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.     

The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1

Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted studies aimed at identifying partner proteins that bind to its C-terminal PDZ ligand motif. Yeast two hybrid studies using the Tech C-terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post-synaptic compartment, as a candidate partner protein for Tech. Co-transfection of Tech and MUPP1 in human embryonic kidney 293 cells confirmed that these full-length proteins interact in a PDZ-dependent fashion. Furthermore, we confirmed that endogenous Tech co-precipitates with MUPP1, but not PSD-95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co-localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. Taken together, these findings demonstrate that Tech binds to MUPP1 and suggest that it regulates RhoA signaling pathways in the vicinity of synapses.     

Control of neurite outgrowth by RhoA inactivation

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.     

LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

LKB1 is a Ser/Thr kinase that plays an important role in controlling both energy metabolism and cell polarity in metazoan organisms. LKB1 is also a tumor suppressor, and homozygous, inactivating mutations are found in a wide range of human cancers. In lung cancer, inactivating mutations are found in 10 to 50% of cases, but the consequences of functional loss in this context are poorly understood. We report here that LKB1 is required for the maturation of apical junctions in the human bronchial epithelial cell line 16HBE14o- (16HBE). This activity is dependent on an interaction with the Rho guanine nucleotide exchange factor p114RhoGEF but is independent of LKB1 kinase activity. Together, LKB1 and p114RhoGEF control RhoA activity in these cells to promote apical junction assembly.     

Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis

Angiogenesis requires concomitant remodeling of cell junctions and migration, as exemplified by recent observations of extensive endothelial cell movement along growing blood vessels. We report that a protein complex that regulates cell junctions is required for VEGF-driven directional migration and for angiogenesis in vivo. The complex consists of RhoA and Syx, a RhoA guanine exchange factor cross-linked by the Crumbs polarity protein Mupp1 to angiomotin, a phosphatidylinositol-binding protein. The Syx-associated complex translocates to the leading edge of migrating cells by membrane trafficking that requires the tight junction recycling GTPase Rab13. In turn, Rab13 associates with Grb2, targeting Syx and RhoA to Tyr(1175)-phosphorylated VEGFR2 at the leading edge. Rab13 knockdown in zebrafish impeded sprouting of intersegmental vessels and diminished the directionality of their tip cells. These results indicate that endothelial cell mobility in sprouting vessels is facilitated by shuttling the same protein complex from disassembling junctions to the leading edges of cells.