Rab13 regulates PKA signaling during tight junction assembly

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell-cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.     

Rac1, but not RhoA,signaling protects epithelial adherens junction assembly during ATP depletion

Rhofamily GTPase signaling regulates actin cytoskeleton and junctionalcomplex assembly. Our previous work showed that RhoA signaling protectstight junctions from damage during ATP depletion. Here, we examinedwhether RhoA GTPase signaling protects adherens junction assemblyduring ATP depletion. Despite specific RhoA signaling- and ATPdepletion-induced effects on adherens junction assembly, RhoA signalingdid not alter adherens junction disassembly rates during ATP depletion.This shows that RhoA signaling specifically protects tight junctionsfrom damage during ATP depletion. Rac1 GTPase signaling also regulatesadherens junction assembly and therefore may regulate adherens junctionassembly during ATP depletion. Indeed, we found that Rac1 signalingprotects adherens junctions from damage during ATP depletion. Adherensjunctions are regulated by various GTPases, including RhoA and Rac1,but adherens junctions are specifically protected by Rac1 signaling.

     

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.     

Tyrosine phosphorylated Par3 regulates epithelial tight junction assembly promoted by EGFR signaling

The conserved polarity complex, comprising the partitioning-defective (Par) proteins Par3 and Par6, and the atypical protein kinase C, functions in various cell-polarization events and asymmetric cell divisions. However, little is known about whether and how external stimuli-induced signals may regulate Par3 function in epithelial cell polarity. Here, we found that Par3 was tyrosine phosphorylated through phosphoproteomic profiling of pervanadate-induced phosphotyrosine proteins. We also demonstrated that the tyrosine phosphorylation event induced by multiple growth factors including epidermal growth factor (EGF) was dependent on activation of Src family kinase (SFK) members c-Src and c-Yes. The tyrosine residue 1127 (Y1127) of Par3 was identified as the major EGF-induced phosphorylation site. Moreover, we found that Y1127 phosphorylation reduced the association of Par3 with LIM kinase 2 (LIMK2), thus enabling LIMK2 to regulate cofilin phosphorylation dynamics. Substitution of Y1127 for phenylalanine impaired the EGF-induced Par3 and LIMK2 dissociation and delayed epithelial tight junction (TJ) assembly considerably. Collectively, these data suggest a novel, phosphotyrosine-dependent fine-tuning mechanism of Par3 in epithelial TJ assembly controlled by the EGF receptor-SFK signaling pathway.     

Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery

Actincytoskeletal disruption is a hallmark of ischemic injury and ATPdepletion in a number of cell types, including renal epithelial cells.We manipulated Rho GTPase signaling by transfection and microinjectionin LLC-PK proximal tubule epithelial cells and observed actincytoskeletal organization following ATP depletion or recovery byconfocal microscopy and quantitative image analysis. ATP depletionresulted in disruption of stress fibers, cortical F-actin, and apicalactin bundles. Constitutively active RhoV14 prevented disruption ofstress fibers and cortical F-actin during ATP depletion and enhancedthe rate of stress fiber reassembly during recovery. Conversely, theRho inhibitor C3 or dominant negative RhoN19 prevented recovery ofF-actin assemblies upon repletion. Actin bundles in the apicalmicrovilli and cytosolic F-actin were not affected by Rho signaling.Assembly of vinculin and paxillin into focal adhesions was disrupted byATP depletion, and constitutively active RhoV14, although protectingstress fibers from disassembly, did not prevent dispersion of vinculinand paxillin, resulting in uncoupling of stress fiber and focaladhesion assembly. We propose that ATP depletion causes Rhoinactivation during ischemia and that recovery of normalcellular architecture and function requires Rho.

     

aPKC-PAR complex dysfunction and tight junction disassembly in renal epithelial cells during ATP depletion

Renal ischemia and in vitro ATP depletion result in disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity. Tight junction formation is regulated by evolutionarily conserved complexes, including that of atypical protein kinase C (aPKC), Par3, and Par6. The aPKC signaling complex is activated by Rac and regulated by protein phosphorylation and associations with other tight junction regulatory proteins, for example, mLgl. In this study, we examined the role of aPKC signaling complex during ATP depletion and recovery in Madin-Darby canine kidney cells. ATP depletion reduced Rac GTPase activity and induced Par3, aPKC, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKC with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKC and Rac activities were suppressed. Experimental inhibition of aPKC during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery. ischemia; atypical PKC; Par3; zonula occludens-1; mLgl-1     

Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis

Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis "decision." Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.     

Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.     

SHP-2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.     

AlphaPIX Rho GTPase guanine nucleotide exchange factor regulates lymphocyte functions and antigen receptor signaling

AlphaPIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletal-membrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of alphaPIX knockout mice. We extended alphaPIX expression experiments and found that whereas alphaPIX was specific to immune cells, its homolog betaPIX was expressed in a wider range of cells. Mice lacking alphaPIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of alphaPIX(-) T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of alphaPIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for alphaPIX in the immune system and suggest that redundancy with betaPIX precludes a more severe immune phenotype.     

Small GTPase Rho regulates thrombin-induced platelet aggregation

Platelets play essential roles in hemostasis and thrombosis by aggregating with each other. However, the molecular mechanism governing platelet aggregation is not yet fully understood. Here, we established an assay system using platelets permeabilized with streptolysin-O to analyze mechanism of the thrombin-induced aggregation, focusing upon a controversial issue in the field whether small GTPase Rho regulates the aggregation. Incubation of the permeabilized platelets with Rho GDP-dissociation inhibitor, an inhibitory regulator for Rho family GTPases, extracted Rho family proteins extensively from the plasma and intracellular membranes, and inhibited the thrombin-induced aggregation. Incubation of the permeabilized platelets with botulinum exoenzyme C3, which specifically inhibits Rho function by ADP-ribosylating it, abolished the thrombin-induced aggregation. Thus, Rho is involved in thrombin-induced aggregation of platelets.     

Structural basis of the Rho GTPase signaling

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.     

Expression and distribution of symplekin regulates the assembly and function of the epithelial tight junction

Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Functions of symplekin at tight junctions have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HT-29 and HepG2 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps to maintain the integrity of the epithelial monolayer and cellular polarity.     

The atypical Rho GTPase,RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.     

Rho GTPase signaling in the development of colorectal cancer

The involvement of Rho GTPases in major aspects of cancer development, such as cell proliferation, apoptosis, cell polarity, adhesion, migration, and invasion, have recently been attracting increasing attention. In this review, we have summarized the current findings in the literature, and we discuss the participation of the Rho GTPase members RhoA, Rac1, and Cdc42 in the development of colorectal cancer, the second most lethal neoplasia worldwide. First, we present an overview of the mechanisms of Rho GTPase regulation and the impact that regulator proteins exert on GTPase signaling. Second, we focus on the participation of Rho GTPases as modulators of colorectal cancer development. Third, we emphasize the involvement of activation and expression alterations of Rho GTPases in events associated with cancer progression, such as loss of cell-cell adhesion, proliferation, migration, and invasion. Finally, we highlight the potential use of novel anticancer drugs targeting specific components of the Rho GTPase signaling pathway with antineoplastic activity in this cancer type.