NGF-induced moesin phosphorylation is mediated by the PI3K,Rac1 and Akt and required for neurite formation in PC12 cells

Moesin is a member of ERM family proteins which act as the cross-linkers between plasma membrane and actin-cytoskeleton and is activated by phosphorylation at Thr-558. In neurons, suppression of radixin and moesin alters the growth cone morphology. However, the significance of phosphorylation of ERM proteins in neuronal cells has not been fully investigated. In this study, we studied the signaling pathways responsible for moesin phosphorylation and its functional importance in NGF-treated PC12 cells. NGF rapidly induced the phosphorylation of moesin at Thr-558 in PC12 cells which was dependent on PI3K and Rac1. We found that Akt interacted and phosphorylated with moesin both in vitro and in vivo. Inhibition of PI3K and Rac1 abolished the NGF-induced Akt activation, indicating that Akt is at the downstream of PI3K and Rac1. To examine the functional role of phosphorylated ERM proteins, a dominant negative mutant form of moesin (T558A) was introduced into PC12 cells. The mutant significantly reduced the frequency of cells with neurites following NGF treatment. Our results indicate that PI3K, Rac1 and Akt-dependent phosphorylation of moesin is required for the NGF-induced neurite formation in differentiating PC12 cells.     

RhoA and Rho kinase-dependent phosphorylation of moesin at Thr-558 in hippocampal neuronal cells by glutamate

When we were studying phosphorylated proteins in the rat brain after electroconvulsive shock (ECS), we observed the rapid phosphorylation of a 75-kDa protein, which cross-reacted with the anti-phospho-p70 S6 kinase antibody. The phosphorylated protein was purified and identified as moesin, a member of the ezrin/radixin/moesin (ERM) family and a general cross-linker between cortical actin filaments and plasma membranes. The purified moesin from rat brain was phosphorylated at serine and threonine residues. Moesin was rapidly phosphorylated at the threonine 558 residue after ECS in the rat hippocampus, peaked at 1 min, and returned to the basal level by 2 min after ECS. To investigate the mechanism of moesin phosphorylation in neuronal cells, we stimulated a rat hippocampal progenitor cell, H19-7/IGF-IR, with glutamate, and observed the increased phosphorylation of moesin at Thr-558. Glutamate transiently activated RhoA, and constitutively active RhoA increased the basal level phosphorylation of moesin. The inhibition of RhoA and its effector, Rho kinase, abolished increased Thr-558 phosphorylation by glutamate in H19-7/IGF-IR cells, suggesting that the phosphorylation of moesin at Thr-558 in H19-7/IGF-IR cells by glutamate is mediated by RhoA and Rho kinase activation.     

RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

Background

The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.

Methods

Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT^558A and pcDNA3/HA-moesinT^558D were applied in endothelial cells.

Results

The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.

Conclusion

These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.     

Age-related changes in lck-Vav signaling pathways in mouse CD4 T cells

Activation of lck-fyn kinases during T cell receptor signaling leads to Vav phosphorylation, activation of downstream targets including Rac1, and a transient decline in ezrin and moesin phosphorylation. We have shown that age increases Rac1 activity and lowers ezrin and moesin phosphorylation in resting mouse CD4 cells, changes that could be the results of alterations in lck-Vav signaling. Analysis of Vav in CD4 cells from old mice shows increases in the phosphorylation of two key regulatory residues, Tyr160 and Tyr174, suggesting enhancement of Vav GTPase activity. In addition, analysis of lck status also shows age-related increases in phosphorylation of two key residues, Tyr394 and Tyr505, which have opposite effects on lck function. These changes in lck-Vav signals in resting CD4 cells may contribute in turn to age-related increases in Rac1 activity and declines in phosphorylation of cytoskeletal proteins including Ezrin and Moesin.     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

Identification and functional implication of a Rho kinase-dependent moesin-EBP50 interaction in noradrenaline-stimulated artery

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.     

Nerve growth factor signals through TrkA,phosphatidylinositol 3-kinase,and Rac1 to inactivate RhoA during the initiation of neuronal differentiation of PC12 cells

In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.     

Role of Moesin in Renal Fibrosis

Background

Renal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.

Method

Human proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.

Result

HK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.

Conclusion

We find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin.     

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.     

Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement

Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

Involvement of Rho GTPases and Their Effectors in the Secretory Process of PC12 Cells

We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K⁺ concentrations was enhanced by the dominant negative mutants RhoA^N19, Rac1^N17, and Cdc42^N17 and was diminished by the constitutively active mutants RhoA^V14, Rac1^V12, and Cdc42^V12. The inhibition observed in the presence of RhoA^V14 was likely a result of the activation of ROKα, since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1^V12 may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K⁺-stimulated exocytosis, and a point mutation in the effector domain of Rac1^V12 that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42^V12 target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.     

Phosphorylation of STEF/Tiam2 by protein kinase A is critical for Rac1 activation and neurite outgrowth in dibutyryl cAMP-treated PC12D cells

The second messenger cAMP plays a pivotal role in neurite/axon growth and guidance, but its downstream pathways leading to the regulation of Rho GTPases, centrally implicated in neuronal morphogenesis, remain elusive. We examined spatiotemporal changes in Rac1 and Cdc42 activity and phosphatidylinositol 3,4,5-triphosphate (PIP₃) concentration in dibutyryl cAMP (dbcAMP)-treated PC12D cells using Förster resonance energy transfer–based biosensors. During a 30-min incubation with dbcAMP, Rac1 activity gradually increased throughout the cells and remained at its maximal level. There was no change in PIP₃ concentration. After a 5-h incubation with dbcAMP, Rac1 and Cdc42 were activated at the protruding tips of neurites without PIP₃ accumulation. dbcAMP-induced Rac1 activation was principally mediated by protein kinase A (PKA) and Sif- and Tiam1-like exchange factor (STEF)/Tiam2. STEF depletion drastically reduced dbcAMP-induced neurite outgrowth. PKA phosphorylates STEF at three residues (Thr-749, Ser-782, Ser-1562); Thr-749 phosphorylation was critical for dbcAMP-induced Rac1 activation and neurite extension. During dbcAMP-induced neurite outgrowth, PKA activation at the plasma membrane became localized to neurite tips; this localization may contribute to local Rac1 activation at the same neurite tips. Considering the critical role of Rac1 in neuronal morphogenesis, the PKA—STEF–Rac1 pathway may play a crucial role in cytoskeletal regulation during neurite/axon outgrowth and guidance, which depend on cAMP signals.     

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) G?6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance.     

Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion

Background

Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted.

Methodology/Principal Findings

In estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17β-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ERα with the G protein Gα₁₃, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The Gα₁₃/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane.

Conclusions/Significance

These results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear Gα₁₃/RhoA/ROCK/moesin signaling cascade as a target of ERα in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.     

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.     

Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac,ADAM17/TACE,and RhoA in tubular epithelial cells

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α–induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.