RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

cGMP-dependent protein kinase phosphorylates and inactivates RhoA

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.     

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (¹⁸⁸S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (¹⁰⁸T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on ⁸⁸S and ¹⁰⁰T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on ⁸⁸S and ¹⁰⁰T in response to EGF, which upregulates RhoA activity.     

PAK1 negatively regulates the activity of the Rho exchange factor NET1

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.     

Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex

RhoA, -B, and -C are ADP-ribosylated by Clostridium botulinum exoenzyme C3 to induce redistribution of the actin filaments in intact cells, a finding that has led to the notion that the ADP-ribosylation blocks coupling of Rho to the downstream effectors. ADP-ribosylation, however, does not alter nucleotide binding, intrinsic, and GTPase-activating protein-stimulated GTPase activity. ADP-ribosylated Rho is even capable of activating the effector protein ROK in a recombinant system. Treatment of cells with a cell-permeable chimeric C3 toxin led to complete localization of modified Rho to the cytosolic fraction based on the complexation of ADP-ribosylated Rho with the guanine-nucleotide dissociation inhibitor-1 (GDI-1). The modified complex turned out to be resistant to phosphatidylinositol 4,5-bisphosphate- and GTPgammaS-induced release of Rho from GDI-1. Thus, ADP-ribosylation leads to entrapment of Rho in the GDI-1 complex. The increased stability of the GDI complex prevented binding of Rho to membrane-associated players of the GTPase cycle such as the activating guanine nucleotide exchange factors and effector proteins.     

Tensins are versatile regulators of Rho GTPase signalling and cell adhesion

Tensins are focal adhesion molecules that were identified and characterised in the late 1980s to early 1990s. They play an essential role in the control of cell adhesion. Tensins can bind the tail of ß integrin via their phospho tyrosine binding domain, they exhibit various protein interaction domains including a Src Homology 2 domain and they are serine‐, threonine‐ and tyrosine‐phosphorylated in response to various stimuli. Tensins serve as scaffolds to gather signalling molecules at the extracellular matrix adhesion complexes. Tensins have emerged as important regulators of cell adhesion and migration, in particular by participating in Rho GTPase signalling pathways. Tensins were shown to influence the activity of the GTPase RhoA, by regulating the Rho GTPase activating protein Deleted in Liver Cancer 1. More recently, Tensin 3 was also found to regulate Dock5, a guanine nucleotide exchange factor for the GTPase Rac, and to modulate podosome‐based adhesion structures in osteoclasts. This review focusses on the recent literature highlighting how Tensins can interplay with regulators of Rho GTPase signalling pathways and how this influences cell adhesion and migration.     

Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization.

It is generally believed that the induction of actin cytoskeleton rearrangements by extracellular stimuli results from the activation of guanine nucleotide exchange factors for the Rho GTPases. Here, we present evidence that the inactivation of RhoGAP (GTPase activating protein) activity is an equally effective means of promoting Rho-mediated cellular processes. We observed that exposure of cultured fibroblasts to sodium fluoride (NaF) results in a rapid and potent stimulation of actin stress fiber formation. This effect is mediated by the Rho GTPase and is associated with the inactivation of cellular RhoGAP activity. Specifically, NaF promotes formation of a high-affinity complex between Rho and the two cellular p190 RhoGAPs in vivo, apparently sequestering limiting amounts of RhoGAP activity, thereby resulting in Rho activation. p190 RhoGAP activity was found to account for approximately 60% of the total RhoGAP activity detected in whole cell extracts, indicating that relatively small changes in cellular RhoGAP activity can have potent effects on Rho activation. We also found that sub-effective concentrations of NaF combined with sub-effective concentrations of the Rho pathway activator, lysophosphatidic acid, which stimulates guanine nucleotide exchange activity on the Rho GTPase, results in the rapid induction of actin stress fibers. Together, these results suggest that the Rho GTPase is regulated by a fine balance of nucleotide exchange and RhoGAP activities, and that inactivation of RhoGAP activity may be a physiologically important regulatory mechanism for activating the Rho GTPase.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.     

Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.

We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.     

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.     

A dual functional signal mediator showing RhoGAP and phospholipase C-delta stimulating activities.

We have cloned a novel regulator protein, p122, in the PLC-delta signalling pathway by screening a rat brain expression library with antiserum raised against purified phospholipase C-delta 1 (PLC-delta 1). This novel p122-RhoGAP binds to PLC-delta 1 and activates the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-delta 1. As suggested by the deduced amino acid sequence, this regulator protein shows a similarity to the GTPase activating protein (GAP) homology region of Bcr and possesses GAP activity for RhoA, but not for Rac1; no guanine nucleotide exchange activity for RhoA and Rac1 was detected. These findings suggest that this novel RhoGAP is involved in the Rho signalling pathway, probably downstream of Rho activation, and mediates the stimulation of PLC-delta, which leads to actin-related cytoskeletal changes through the hydrolysis of PIP2, which binds to actin binding proteins such as gelsolin and profilin.     

Regulatory models of RhoA suppression by dematin,a cytoskeletal adaptor protein

Cell motility, adhesion, and actin cytoskeletal rearrangements occur upon integrin-engagement to the extracellular matrix and activation of the small family of Rho GTPases, RhoA, Rac1, and Cdc42. The activity of the GTPases is regulated through associations with guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine dissociation inhibitors (GDIs). Recent studies have demonstrated a critical role for actin-binding proteins, such as ezrin, radixin, and moesin (ERM), in modulating the activity of small GTPases through their direct associations with GEFs, GAPs, and GDI’s. Dematin, an actin binding and bundling phospho-protein was first identified and characterized from the erythrocyte membrane, and has recently been implicated in regulating cell motility, adhesion, and morphology by suppressing RhoA activation in mouse embryonic fibroblasts. Although the precise mechanism of RhoA suppression by dematin is unclear, several plausible and hypothetical models can be invoked. Dematin may bind and inhibit GEF activity, form an inactive complex with GDI-RhoA-GDP, or enhance GAP function. Dematin is the first actin-binding protein identified from the erythrocyte membrane that participates in GTPase signaling, and its broad expression suggests a conserved function in multiple tissues.     

Regulation of ephexin1, a guanine nucleotide exchange factor of Rho family GTPases, by fibroblast growth factor receptor-mediated tyrosine phosphorylation

Fibroblast growth factor (FGF) signal is implicated in not only cell proliferation, but cell migration and morphological changes. Several different Rho family GTPases downstream of the Ras/ERK pathway are postulated to mediate the latter functions. However, none have been recognized to be directly coupled to FGF receptors (FGFRs). We have previously reported that EphA4 and FGFRs hetero-oligomerize through their cytoplasmic domains, trans-activate each other, and transduce a signal for cell proliferation through a docking protein, FRS2alpha (Yokote, H., Fujita, K., Jing, X., Sawada, T., Liang, S., Yao, L., Yan, X., Zhang, Y., Schlessinger, J., and Sakaguchi, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 18866-18871). Here, we have found that ephexin1, a guanine nucleotide exchange factor for Rho family GTPases, constitutes another downstream component of the receptor complex. Ephexin1 directly binds to the kinase domain of FGFR mainly through its DH and PH domains. The binding appears to become weaker and limited to the DH domain when FGFRs become activated. FGFR-mediated phosphorylation of ephexin1 enhances the guanine nucleotide exchange activity toward RhoA without affecting the activity to Rac1 or Cdc42. The FGFR-mediated tyrosine phosphorylation includes, but is not limited to, the residue (Tyr-87) phosphorylated by Src family kinase, which is known to be activated following EphA4 activation. The Tyr-to-Asp mutations that mimic the tyrosine phosphorylation in some of the putative FGFR-mediated phosphorylation sites increase the nucleotide exchange activity for RhoA without changing the activity for Rac1 or Cdc42. From these results, we conclude that ephexin1 is located immediately downstream of the EphA4-FGFR complex and the function is altered by the FGFR-mediated tyrosine phosphorylation at multiple sites.     

GDIs: central regulatory molecules in Rho GTPase activation

The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.     

Rho and Rac take center stage

Many features of cell behavior are regulated by Rho family GTPases, but the most profound effects of these proteins are on the actin cytoskeleton and it was these that first drew attention to this family of signaling proteins. Focusing on Rho and Rac, we will discuss how their effectors regulate the actin cytoskeleton. We will describe how the activity of Rho proteins is regulated downstream from growth factor receptors and cell adhesion molecules by guanine nucleotide exchange factors and GTPase activating proteins. Additionally, we will discuss how there is signaling crosstalk between family members and how various bacterial pathogens have developed strategies to manipulate Rho protein activity so as to enhance their own survival.     

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.     

IgA1 protease from Neisseria gonorrhoeae inhibits TNFalpha-mediated apoptosis of human monocytic cells

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.     

BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity

Myosin IXb, a member of the myosin superfamily, is a molecular motor that possesses a GTPase activating protein (GAP) for Rho. Through the yeast two-hybrid screening using the tail domain of myosin IXb as bait we found BIG1, a guanine nucleotide exchange factor for ADP-ribosylation factor (Arf1), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced, and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by the addition of BIG1 with IC(50) of 0.06 microm. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with the concentration similar to the inhibition of the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the Arf guanine nucleotide exchange factor, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule.