Table of contents

Signaling downstream from Rho and Rho effects on the cytoskeleton

In Saccharomyces cerevisiae, the phosphatidylinositol kinase homolog Tor2 controls the cell-cycle-dependent organization of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2. Four Rho1 effectors are known: protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signaling protein Skn7. It has been suggested that Rho1 signals to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organization, however. The role of Rho1 in controlling actin organisation has been investigated and analysed as well as which of the Rho1 effectors mediates Tor2 signaling to the actin cytoskeleton was anayzed. Some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganized actin cytoskeleton. Both the growth defect and the actin organization defect of the rho1-2ts mutant are suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7. Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescues a tor2ts mutant, and deletion of BNI1 or SKN7 does not prevent the suppression of the tor2ts mutation by overexpressed Rom2. Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppresses the actin defect of tor2ts and rho1-2ts mutants. Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade (Helliwell, 1998).

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 (see Drosophila LIM-kinase1) and kinase-dead LIMK2 (LIMK2/KD) are respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylates cofilin and induces formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation is stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induces stress fibers and filopodia, whereas both Rho-induced stress fibers and Cdc42-induce filopodia are abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac does not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization (Sumi, 1999).

The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. RGD1 acts somewhere upstream of the PKC pathway. Given that Rgd1p has been shown to have a Rho-GAP activity toward Rho3p and Rho4p, the defect of PKC pathway activation at late exponential phase observed in the rgd1 mutants might be mediated by the small GTPases Rho3p and Rho4p. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta, RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog were each shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. It is hypothesized that rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway (de Bettignies, 2001).

In Saccharomyces cerevisiae, the Rho family of GTPases is thought to have a central role in the polarized growth process. The main functions assigned to these GTPases involve bud formation and cell surface growth, which might occur through the involvement of the actin cytoskeleton and the secretory pathway. Genetic and functional analyses have allowed the identification of five Rho members in yeast: Cdc42 and Rho1 to Rho4. These small GTPases function as binary switches, which are turned on and off by binding to GTP or GDP, respectively. The GTP-bound form interacts with its specific target and performs its cell functions. Small GTPases are regulated by GAPs (GTPase-activating proteins), GEFs (GDP-GTP exchange factors), and a GDP dissociation inhibitor (de Bettignies, 2001).

During the sequencing of the genome of S. cerevisiae, a new gene encoding a protein with a Rho-GAP homology domain was identified. This protein, called Rgd1p (for related GAP domain), was shown in vitro to be a GTPase activating protein for the Rho3 and Rho4 proteins. Thus, in activating the hydrolysis of GTP, Rgd1p negatively regulates the action of these two Rho proteins. Rho3p and Rho4p play a role in bud formation and have some partially overlapping functions. Deletion of RHO4 does not affect cell growth, whereas deletion of RHO3 causes a severe growth delay and a decrease in cell viability. Overexpression of RHO4 suppresses the growth defect in rho3 cells. Depletion of both RHO3 and RHO4 gene products results in lysis of cells with a small bud, which can be prevented by the presence of osmotic stabilizer in the medium. In this latter condition, Rho3p- and Rho4p-depleted cells lose cell polarity as revealed by chitin delocalization and by random distribution of actin patches (de Bettignies, 2001).

Genetic interactions occur between RGD1 and the SLG1 and MID2 genes. SLG1 has also been designated HCS77 and WSC1, but for simplicity this gene is referred to here as SLG1. Slg1p and Mid2p are both plasma membrane proteins with partial overlapping functions. They act upstream of the protein kinase C (PKC) pathway and are thought to monitor the state of the cell surface and relay the information to Pkc1p. Protein kinase C is mostly regulated by the small GTPase Rho1p in vivo. Pkc1p activates a mitogen-activated protein (MAP) kinase cascade, named the PKC pathway, consisting of Bck1p, Mkk1p/Mkk2p, and the MAP kinase Mpk1p. Activation of this pathway is particularly important in response to various external stresses, including high temperature, low osmolarity, and cell wall disruption, as well as being important during mating. The protein Slg1 is linked to the PKC pathway by the finding that this MAP kinase cascade is activated by heat stress via Slg1p. A direct interaction of Slg1p with Rom2p, one of the Rho1p-GEFs, has been recently reported and this interaction is responsible for the activation of the PKC pathway through Rho1p (de Bettignies, 2001).

The loss of RGD1 function amplifies the phenotype due to the SLG1 deletion and the small-budded double-mutant cells die because of defects in cell wall structure and lysis upon bud growth. In parallel, the inactivation of MID2, the other putative sensor for cell integrity signaling in S. cerevisiae, exacerbates the specific phenotype of the rgd1Delta mutant with an increase in dead cells at late exponential phase in minimal medium. Taken together, these results suggest that Rgd1p has a regulatory role in connection with both the PKC pathway and the actin cytoskeleton organization in S. cerevisiae (de Bettignies, 2001).

To further elucidate the function of RGD1, multicopy suppressors of the viability defect of the rgd1Delta mutation were isolated in minimal medium. Phenotypic and genetic analysis has allowed the identification of several multicopy as well as monocopy suppressors of rgd1Delta: the RHO1 and RHO2 genes encoding two GTPases involved in actin cytoskeleton organization, the MID2-homolog MTL1, and the MKK1 gene coding for one of the MAP-kinase kinases of the PKC pathway. Considering the suppressor effect of additional PKC pathway components, it has been shown that activation of the PKC pathway prevents lethality of rgd1Delta cells. Analysis of the transcriptional activity of Rlm1p, one of the targets of the last kinase in the PKC pathway, and study of the PST1 transcription, which is positively regulated by Rlm1p, shows that the rgd1Delta mutation decreases the activity of this MAP-kinase pathway in minimal medium at late exponential phase. This decrease in PKC pathway activity is at least partly responsible for the rgd1Delta cell viability loss under particular growth or physiological conditions (de Bettignies, 2001).

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that have been 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 for this regulation are unclear. Lysophosphatidic acid stimulation of serum-starved NIH3T3 cells results in relocalization of radixin into apical membrane/actin protrusions: this relocation is blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, is sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment leads to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin reveals that RhoA activity is necessary and sufficient for their phosphorylation. Growth factors may mediate the localization of the ERM proteins via a RhoA-dependent kinase, such as protein kinase N, Rho-kinase, or PRK. These findings reveal a novel function for RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins (Shaw, 1998).

The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacts with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylates MBS and consequently inactivates myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increases phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase (Kimura, 1996).

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation are decreased, and cell spreading is inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation (Sanders, 1999).

The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor - See Drosophila Blistered) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for the regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway (Hill, 1995).

RhoA and two other Rho-family proteins, Cdc42 and Rac1, regulate Serum Response Factor (SRF) activation of the c-fos serum response element. This pathway acts independently of known MAPK pathways and is regulated by agents such as serum and LPA, acting via heterotrimeric G protein-coupled receptors. Constitutively active forms of either of the small GTPases -- RhoA (RhoA.V14) or Cdc42 (Cdc42.V12) -- induces expression of extrachromosomal SRF reporter genes in microinjection experiments, but only Cdc42.V12 can efficiently activate a chromosomal template. Both SAPK/JNK-dependent or -independent signals can cooperate with RhoA.V14 to activate chromosomal SRF reporters; it is SAPK/JNK activation by Cdc42.V12 that allows SAPK/JNK to activate chromosomal templates. Cooperating signals can be bypassed by deacetylase inhibitors. Three findings show that histone H4 hyperacetylation is one target for cooperating signals, although it alone is not sufficient: (1) Cdc42.V12, but not RhoA.V14, induces H4 hyperacetylation; (2) cooperating signals use the same SAPK/JNK-dependent or -independent pathways to induce H4 hyperacetylation, and (3) growth factor and stress stimuli induce substantial H4 hyperacetylation, detectable in reporter gene chromatin. These data establish a link between signal-regulated acetylation events and gene transcription. Thus, in isolation, the SRF-controlled extrachromosomal reporter gene is a target for only a subset of signals that can activate the chromsomal c-fos promoter. This is thought to reflect differences in chromatin structure associated with the two types of templates (Alberts, 1998a).

The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. Overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-kappaB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, because a dominant-negative MEK and a MEK inhibitor (PD98059) do not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Calpha (PKCalpha) in vivo; however, deletion of the CAAX site on V14Rho severely diminishes this association. Evidence for a role for PKCalpha as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCalpha blocks the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation (Chang, 1998).

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation: the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signaling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signaling pathways to varying degrees. Other signaling pathways could also influence the consequences of Ras signaling. When signaling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Therefore, Rho helps Ras to drive cells into S phase. Cells that lack p21Waf1/Cip1 do not require Rho signaling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signaling is the suppression of p21Waf1/Cip1 induction (Olson, 1998).

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. A similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supports growth factor-induced, E2F-dependent S phase entry. Although all substrates support the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity is specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurs specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression (Danen, 2000).

The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization, transcription and transformation. Although many different potential RhoA effectors have been identified, including two families of protein kinases, their roles in RhoA-regulated events remain unclear. A genetic screen was used to identify mutations at positions 37-42 in the RhoA effector loop that selectively disrupt effector binding, and these were used to investigate the role of RhoA effectors in the formation of actin stress fibres, activation of transcription by serum response factor (SRF) and transformation. Interaction with the ROCK kinase and at least one other unidentified effector is required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of RhoA-induced cytoskeletal changes, and does not correlate with binding to any of the effectors tested, indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of SRF, correlates with the activity of RhoA in transformation. The effector mutants should provide novel approaches for the functional study of RhoA and isolation of effector molecules involved in specific signalling processes (Sahai, 1998).

The ubiquitously expressed Na-H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Rho-associated kinase isozymes, p160ROCK and ROKalpha/Rho-kinase/ROCK-II, mediate RhoA-induced assembly of focal adhesions and actin stress fibers. p160ROCK and ROKalpha/Rho-kinase/ROCK-II are coiled-coil-forming serine/threonine kinases sharing ~90% identity within the kinase domain. It is suggested that these kinases regulate cell contractility by indirectly increasing phosphorylation of myosin light chain through the inhibition of myosin phosphatase activity. Stress fiber formation induced by p160ROCK is inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and is absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity is stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibits RhoA-, but not Cdc42- or Rac-activation of NEH1. The p160ROCK-specific inhibitor Y-27632 inhibits increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA. It also inhibits LPA-increased phosphorylation of NHE1. A C-terminal truncation of NHE1 abolishes both LPA-induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylates an NHE1 C-terminal fusion protein in vitro; this is inhibited in the presence of Y-27632. Phosphopeptide maps indicate that identical residues in NHE1 are phosphorylated by p160ROCK in vivo and in vitro. These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber asssembly induced by p160ROCK (Tominaga, 1998).

The specific signal that NHE1 is contributing to regulate Rho-mediated cytoskeletal remodeling remains to be determined. The effects of NHE1 are likely to be mediated by changes in intracellular concentrations of H+ or Na+, or by changes in cell volume. The predominant localization of NHE1 at sites of focal contact suggests that if H+ is an important signal, then perhaps localized pHi gradients might be critical for the assembly of focal adhesions and the focal attachment of actin stress fibers. If localized pHi gradients are an important signal, the current findings suggest that these are generated primarily by NHE1, as HCO3-dependent exchangers are unable to compensate for the loss of NHE1 activity. An alternative possibility is that NHE1 is structurally linked to the actin cytoskeleton, analogous to the role of the erythrocyte Cl-HCO3 exchanger, AE1. AE1 and NHE1 share a similar structural topology: 12 transmembrane domains and a long cytoplasmic domain; they also share a similar function in regulating pHi. AE1, however, also functions to tether actin to the plasma membrane through the binding of its cytoplasmic domain to the actin-associated proteins ankyrin and protein 4.1 (Tominaga, 1998 and references).

Transforming growth factor beta (TGF-beta) is a multifunctional factor that induces a wide variety of cellular processes affecting growth and differentiation. TGF-beta exerts its effects through a heteromeric complex between two transmembrane serine/threonine kinase receptors, the type I and type II receptors. However, the intracellular signaling pathways through which TGF-beta receptors act to generate cellular responses remain largely undefined. TGF-beta initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. TGF-beta-mediated signaling is abolished by the expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways, including Rho-like GTPases, mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun.Therefore, the SAPK/JNK activation contributes to TGF-beta signaling (Atfi, 1997).

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. Serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. An investigation was carried out to determine if SRF is also involved in the control of Myf5 gene expression, and to assess the potential role of upstream regulators of SRF activity (the Rho family G-proteins, including Rho, Rac, and CDC42) in the regulation of MyoD and Myf5. Inactivation of SRF does not alter Myf5 gene expression, but it does cause a rapid extinction of MyoD gene expression. RhoA (but not Rac or CDC42) is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, results in a dramatic decrease of MyoD protein levels and promoter activity without any effect on Myf5 expression. RhoA-dependent transcriptional activation requires functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, these results establish the first link between RhoA protein activity and the expression of a key muscle regulator (Carnac, 1998).

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. Lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho it has been shown that rho is both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induces the stabilization of only a subset of the MTs: in wound-edge cells, these stable MTs are appropriately oriented toward the leading edge of the cell. LPA has little effect on individual parameters of MT dynamics, but does induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability is independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly (Cook, 1998).

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to first induce disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers show radial-like morphology that is apparently different from the original. The mechanisms of these TPA-induced processes have been analyzed. Rho inactivation and activation are necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibit the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibits the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation is necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions is closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility (Imamura, 1998).

The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, ß-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. A signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing ß-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibit ß-actin mRNA from localizing in response to growth factors. It is concluded that the sorting of ß-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and it is postulated that polarized actin bundles direct the mRNA to the leading edge of the cell (Latham, 2001).

How could myosin II direct mRNA movement to the leading edge? It is postulated that the two-headed myosin filaments can translocate on polarized bundles of actin filaments (a.k.a., 'stress fibers') toward the leading edge associated with an mRNA complex that can bind the myosin. This hypothesis is supported by work demonstrating that, in moving fibroblasts, these actin bundles have a polarity with barbed ends increasingly directed toward the lamellipodium and thus could constrain activated myosin II-B movement only toward the leading edge. There is evidence that myosin activation and deactivation can be spatially regulated. Rho-kinase can lead to phosphorylation of the light chains internally near the nucleus, where myosin filament assembly, stress fiber formation, and motility occurs. PKC in the periphery of the cell can phosphorylate myosin II-B heavy chains and promote disassembly at the leading edge. This could transport the mRNA bound to the myosin toward the leading edge, where it would anchor to actin filaments when the myosin filament disassembles. Extracellular signals can act through the signaling mediators Rho and Rho-kinase in the regulation of ß-actin mRNA distribution via actomyosin interactions. In this way, spatially localized protein synthesis is a component of gene expression that can respond rapidly (2 min) to extracellular signals and immediately effect physiological changes within specific cellular compartments (Latham, 2001).

Studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation. This study demonstrates novel functions for the Rho family GTPases Rac and Rho in the opposing hormonal regulation of voltage-activated, ether-a-go-go-related potassium channels (ERG) in a rat pituitary cell line, GH4C1. The hypothalamic neuropeptide, thyrotropin-releasing hormone (TRH) inhibits ERG channel activity through a PKC-independent process that is blocked by RhoA(19N) and the Clostridium botulinum C3 toxin, which inhibit Rho signaling. The constitutively active, GTPase-deficient mutant of RhoA(63L) rapidly inhibits the channels when the protein is dialysed directly into the cell through the patch pipette, and inhibition persists when the protein is overexpressed. In contrast, GTPase-deficient Rac1(61L) stimulates ERG channel activity. The thyroid hormone triiodothyronine (T3), which antagonizes TRH action in the pituitary, also stimulates ERG channel activity through a rapid process that is blocked by Rac1(17N) and wortmannin but not by RhoA(19N). It is concluded that Rho stimulation by G13-coupled receptors and Rac stimulation by nuclear hormones through PI3-kinase may be general mechanisms for regulating ion channel activity in many cell types. Disruption of these novel signaling cascades is predicted to contribute to several specific human neurological diseases, including epilepsy and deafness (Storey, 2002).

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. A novel and critical role is demonstrated for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models leads to apoptosis and failures in tube formation. A novel link has been identified between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy reveals that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is found throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. This colocalization is functionally relevant, because when RhoB is depleted, Akt is excluded from the nucleus and total cellular Akt protein is decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to be rate limiting only for endothelial cell sprouting, it is proposed that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable (Adini, 2003).

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. The kinase Mirk (minibrain-related kinase: see Drosophila Minibrain )/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase that is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct is activated when C2C12 myoblasts are switched from growth to differentiation medium and is also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increase following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulate Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription is induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk is induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24-48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk is induced following the commitment stage of myogenesis. Stable overexpression of Mirk enables myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevents myoblast fusion into myotubes and inhibits induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development (Deng, 2003 ).

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. Whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization was investigated. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functions as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevents microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, is sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibits fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration (Wen, 2004).

Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. This study describes the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways (Jaffe, 2004).

Rho is a small GTPase that controls signal transduction pathways in response to a large number of extracellular stimuli. With over 15 potential Rho target proteins identified to date, however, it is not clear how distinct signaling outputs can be generated downstream of a particular stimulus. Several of the known Rho targets are structurally reminiscent of scaffold proteins, which are generally thought to play an important role in controlling signaling specificity. The Rho target CNK1 is a scaffold protein that interacts with Net1 or p115RhoGEF, two Rho-specific guanine nucleotide exchange factors (GEFs), as well with MLK2 and MKK7, two of the kinase components in the JNK MAP kinase cascade. CNK1 acts cooperatively with the two GEFs to activate JNK MAP kinase, but not other Rho-mediated pathways. In HeLa cells, serum or sphingosine-1-phosphate stimulate Rho-dependent activation of the JNK MAP kinase cascade, and this requires endogenous CNK1. It is concluded that CNK1 couples a subset of Rho exchange factors to activation of the JNK MAP kinase pathway and that signaling specificity is achieved through complexes containing both upstream activators and downstream targets of Rho (Jaffe, 2005).

Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. This study presents the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its 'switch' regions for interacting with two subdomains of GBD/FH3. The FH3 domain of mDia1 forms a stable dimer, and the DAD-binding site has been identified. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of these results, a structural model for the regulation of mDia1 by Rho and DAD is reported (Rose, 2005).

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. The crystal structure of the dimeric regulatory domain of the DRF, mDia1, is reported in this study. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design (Otomo. 2005).

Connector enhancer of KSR (CNK) proteins have been proposed to act as scaffolds in the Ras-MAPK pathway. In this work, using in vivo bioluminescence resonance energy transfer (BRET) assays and in vitro co-immunoprecipitation, hCNK1 is shown to interact with the active form of Rho A (G14V) proteins. The domain of hCNK1 that allows binding to Rho proteins involves the C-terminal PH domain. Overexpression of hCNK1 does not affect the actin cytoskeleton and does not modify the appearance of stress fibers in cells overexpressing a constitutively active form of RhoA. In contrast, hCNK1 was able to significantly decrease the RhoA-induced transcriptional activity of the serum response element (SRE) without effect on the Ras-induced SRE activation. These results identify hCNK1 as a specific partner of Rho proteins both in vitro and in vivo and suggest a role of hCNK1 in the signal transduction of Rho proteins (Lopez-Ilasaca, 2005).

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-β-catenin signaling pathway requires both Gαo and Gαq heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gαo protein. Gαo propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-β-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-β-catenin signaling (Bikkavilli, 2008).

MKL1/MAL is Rho-regulated co-activator for SRF

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that activates serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, its role in serum induction of c-fos and other SRE-regulated genes was assessed with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). DN-MKL1 and RNAi was found to specifically block SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi requires the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduces the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, is nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia has a markedly increased ability to activate SRE reporter genes, suggesting that fusion protein activation of SRF target genes may contribute to leukemogenesis (Cen, 2003).

Rho GTPases regulate the transcription factor SRF via their ability to induce actin polymerization. SRF activity responds to G actin, but the mechanism of this has remained unclear. Rho-actin signaling has been shown to regulate the subcellular localization of the myocardin-related SRF coactivator MAL, rearranged in t(1;22)(p13;q13) AML. The MAL-SRF interaction displays the predicted properties of a Rho-regulated SRF cofactor. MAL is predominantly cytoplasmic in serum-starved cells, but accumulates in the nucleus following serum stimulation. Activation of the Rho-actin signaling pathway is necessary and sufficient to promote MAL nuclear accumulation. MAL N-terminal sequences, including two RPEL motifs, are required for the response to signaling, while other regions mediate its nuclear export (or cytoplasmic retention) and nuclear import. MAL associates with unpolymerized actin through its RPEL motifs. Constitutively cytoplasmic MAL derivatives interfere with MAL redistribution and Rho-actin signaling to SRF. MAL associates with several SRF target promoters regulated via the Rho-actin pathway (Miralles, 2003).

RhoA signaling regulates the activity of the transcription factor SRF (serum response factor) during muscle differentiation. How RhoA signaling is integrated at SRF target promoters to achieve muscle-lineage-specific expression is largely unknown. Using large-scale expression profiling combined with bioinformatic and biochemical approaches, several SRF target genes were identified, including Fhl2, encoding a transcriptional cofactor that is highly expressed in the heart. SRF binds the Fhl2 promoter in vivo and regulates Fhl2 expression in response to RhoA activation. FHL2 protein and SRF interact physically, and FHL2 binds the promoters of SRF-responsive smooth muscle (SM) genes, but not the promoters of immediate-early genes (IEGs), in response to RhoA. FHL2 antagonizes induction of SM genes, but not IEGs or cardiac genes, by competing with the coactivator MAL/MRTF-A for SRF binding. These findings identify an autoregulatory mechanism to selectively regulate subsets of RhoA-activated SRF target genes (Philippar, 2004).

Myocardin (MC) family proteins are transcriptional coactivators for serum response factor (SRF). Each family member possesses a conserved N-terminal region containing three RPEL motifs (the 'RPEL domain'). MAL/MKL1/myocardin-related transcription factor A is cytoplasmic, accumulating in the nucleus upon activation of Rho GTPase signaling, which alters interactions between G-actin and the RPEL domain. This study demonstrates that MC, which is nuclear, does not shuttle through the cytoplasm and that the contrasting nucleocytoplasmic shuttling properties of MAL and MC are defined by their RPEL domains. The MAL RPEL domain binds actin more avidly than that of MC and that the RPEL motif itself is an actin-binding element. RPEL1 and RPEL2 of MC bind actin weakly compared with those of MAL, while RPEL3 is of comparable and low affinity in the two proteins. Actin binding by all three motifs is required for MAL regulation. The differing behaviors of MAL and MC are specified by the RPEL1-RPEL2 unit, while RPEL3 can be exchanged between them. It is proposed that differential actin occupancy of multiple RPEL motifs regulates nucleocytoplasmic transport and activity of MAL (Guettler, 2008).

Regulation of Rho GTPases by crosstalk

Proper development of neurons depends on synaptic activity, but the mechanisms of activity-dependent neuronal growth are not well understood. The small GTPases, RhoA, Rac, and Cdc42, regulate neuronal morphogenesis by controlling the assembly and stability of the actin cytoskeleton. An in situ method to determine endogenous Rho GTPase activity in intact Xenopus brain is reported. This method was used to provide evidence for crosstalk between Rho GTPases in optic tectal cells. Moreover, crosstalk between the Rho GTPases appears to affect dendritic arbor development in vivo. Optic nerve stimulation regulates Rho GTPase activity in a glutamate receptor-dependent manner. These data suggest a link between glutamate receptor function, Rho GTPase activity, and dendritic arbor growth in the intact animal (Li, 2002).

The inability to assay endogenous Rho GTPase activity in the intact organism has hampered studies of regulation of Rho GTPases. Rho GTPase activity can be determined in cultured cells by pull-down assays in which the extent of the GTP-bound form of Rho GTPase that binds to the specific binding domain of a downstream effector is quantified. This method has been used extensively to detect GTP-bound RhoA, Rac, and Cdc42. The assays take advantage of downstream effectors that only bind the GTP-bound form of the GTPases and that are specific for Rac, RhoA, or Cdc42. For instance, the RhoA binding domain (RBD) of the specific Rho effector Rhotekin, fused to glutathione-S-transferase (GST), has been used to pull down GTP-Rho from cells transfected with constitutively active RhoAV14. PAK is a downstream effector of Rac and Cdc42. The GTPase binding domain of PAK reports Rac and Cdc42 activity. WASP is a downstream effector of Cdc42 but not Rac. Therefore, fusion proteins of the WASP GTPase binding domain with a reporter protein selectively identifies GTP-Cdc42. To report the cellular localization of activated GTPases, fusion proteins of the PAK GTPase binding domain and a fluorescent reporter dye were used to visualize activated Rac in cultured cells by direct fluorescence imaging and fluorescence resonance energy transfer. Similarly, transfected WASP GTPase binding domain and GFP fusion protein was used to show the subcellular distribution of active Cdc42 in living cultured cells. This study uses GST fusion proteins of the GTPase binding domains of PAK, WASP, and Rhotekin to selectively bind endogenous three Xenopus Rho GTPases; respectively Rac, Cdc42, and RhoA (Li, 2002).

Rho GTPases can regulate the activity of other Rho GTPases. RhoA activity is increased by activated Rac and inhibition of Cdc42, while Rac is inhibited by activation of RhoA. Furthermore, the crosstalk observed with the biochemical assay relates to morphological changes in tectal cell dendritic arbor development. Rac regulates dendritic branch dynamics in tectal cells. Exposing animals to LPA, an activator of RhoA, suppresses Rac activity as detected by the in situ assay and prevents Rac-induced increase in branch dynamics. Therefore, data from the in vivo time-lapse imaging and in situ Rho GTPases assay are consistent with the idea that Rho GTPases cooperate to control neuronal morphology. An important question regarding the crosstalk between Rho GTPases concerns the molecular mechanisms of this regulation. In tectal neurons, inhibiting ROK with Y-27632 results in increased dendritic arbor growth rate that can be abolished by expressing dominant-negative Rac; however, Y-27632 does not alter Rac activity. Although this rules out regulation of Rac by ROK, other RhoA effectors, including mDia, could mediate the inhibition of Rac by RhoA. RhoA could also antagonize Rac by competing for common regulatory molecules, such as GEFs (Li, 2002).

Different Rho GTPases are specialized to control different aspects of dendritic growth through their selective effects on the cytoskeleton. RhoA activity inhibits branch extension, whereas Rac promotes branch addition and stabilization. It is suggested that the different GTPases coordinate their activities through crosstalk to control neuronal morphogenesis when stimulated by extracellular signals. Neuronal activity that activates glutamate receptors is one signal that regulates Rho GTPases. Based on this knowledge, a model is proposed for Rho GTPase function in activity-dependent dendritic arbor plasticity. In unstimulated neurons, RhoA activity is high; this maintains low levels of Rac activity through crosstalk. The high RhoA activity and low Rac activity restrict the growth of dendritic arbor so that neurons maintain their structure without further elaboration. Glutamatergic synaptic activity changes Rho GTPase activity: RhoA is inhibited and Rac is activated. The growth of the dendritic arbor is promoted in response to these changes of Rho GTPases activity. Activated Rac enhances branch additions and RhoA inhibition enhances branch elongation. Activated Rac also increases RhoA activity, which in turn may serve to curtail excessive growth in response to synaptic activity. A similar mechanism, based on tight cross regulation of GTPase activities, might be used in other growth factor signaling pathways. Extracellular signals can achieve diverse structural outcomes by differentially regulating Rho GTPases directly and by further modifying the differences in activity levels through crosstalk between Rho GTPases. Therefore, Rho GTPases not only transduce, but also integrate, the growth regulatory signals for neuronal morphogenesis (Li, 2002).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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