Different regulation of the Trio Dbl-Homology domains by their associated PH domains

Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.     

The Human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling.     

The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.     

Catching a GEF by its tail

The activation of Rho GTPases is mediated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP. Rho-GEFs are a very diverse family, with >70 members in humans. Bioinformatics analysis of the human Rho-GEFs shows that approximately 40% contain a putative PDZ-binding motif at the C-terminus. PDZ domains are protein-protein interaction domains that act as scaffolds to concentrate signaling molecules at specialized regions in the cell. We propose that the interaction between Rho-GEFs and PDZ-domain proteins is a general mechanism that controls Rho-GEF targeting and activation, helping to restrict and concentrate the exchange activity to appropriate subcellular destinations. Here, we summarize recent data that highlight the importance of these interactions in Rho-GEF regulation.     

A Highlights from MBoC Selection: Involvement of p114-RhoGEF and Lfc in Wnt-3a– and Dishevelled-Induced RhoA Activation and Neurite Retraction in N1E-115 Mouse Neuroblastoma Cells

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a–induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF–silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a–induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a–induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a– and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.     

The many faces of the guanine-nucleotide exchange factor trio

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.     

The many faces of the guanine-nucleotide exchange factor trio

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.     

Identification of novel neuronal isoforms of the Rho-GEF Trio

BACKGROUND INFORMATION: The large family of GEFs (guanine nucleotide-exchange factors) for Rho GTPases activate the GTPases by accelerating their GDP/GTP exchange. The multidomain protein Trio is the founding member of an intriguing subfamily of Rho-GEFs exhibiting two Rho-GEF and numerous additional domains. The members of the Trio family play an important role in neuronal physiology, and their structural organization is very well conserved through evolution. It has previously been shown that all the members, except mammalian Trio, display several isoforms, the functions of which have been well established. RESULTS: In this study, we have identified, by a combination of different approaches, novel Trio isoforms that have been generated by alternative splicing, giving rise to proteins that exhibit one or two Rho-GEF domains (GEFDs). These isoforms are specifically expressed in the nervous system, at a higher level than the full-length Trio, which is ubiquitously expressed. In addition, we show that all the GEFD1-containing isoforms induce neurite outgrowth in neuroblastoma cells. CONCLUSIONS: We have identified neuronal specific isoforms of Trio which could be essential for Trio function in neuronal morphology.     

Rho-specific Guanine nucleotide exchange factors (Rho-GEFs) inhibition affects macrophage phenotype and disrupts Golgi complex

Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.     

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.     

Regulation of Immature Dendritic Cell Migration by RhoA Guanine Nucleotide Exchange Factor Arhgef5

There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gβγ interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1α-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.Leukocyte chemotaxis underlies leukocyte migration, infiltration, trafficking, and homing that are not only important for normal leukocyte functions, but also have a important role in inflammation-related diseases. Leukocyte chemotaxis is regulated by leukocyte chemoattractants that include bacterial by-products such as formylmethionylleucylphenylalanine, complement proteolytic fragments such as C5a, and the superfamily of chemotactic cytokines, chemokines. These chemoattractants bind to their specific cell G protein-coupled receptors and are primarily coupled to the G_i family of G proteins to regulate leukocyte chemotaxis. Previous studies have established that the Rho family of small GTPases regulates leukocyte migration (1, 2). Rac, Cdc42, and RhoA are the three best studied Rho small GTPases. In myeloid cells, Cdc42 regulates directionality by directing where F-actin and lamellipodia are formed, and Rac regulates F-actin formation in the lamellipodia, which provides a driving force for cell motility (3–6). On the other hand, RhoA regulates the formation and contractility of the actomyosin structure at the back that provides a pushing force (5, 7). Rho guanine nucleotide exchange factors (GEF)³ are key regulators for the activity of these small GTPases. GEFs activate small GTPases by promoting the loading of GTP to the small GTPases, a rate-limiting step in GTPase regulation (8–11). Previous biochemical and genetic studies have revealed how Cdc42 and Rac may be regulated by chemokine receptors in leukocytes. Chemokine receptors can regulate Cdc42 via a Rho-GEF PIXα, which is regulated by Gβγ from the G_i proteins via the interactions between Gβγ and Pak1 and between Pak1 and PIXα in myeloid cells 12. On the other hand, in neutrophils chemokine receptors regulate Rac2 via another Rho-GEF P-Rex1, which is directly regulated by Gβγ (13–15). Two Rho-GEFs have been implicated in regulation of RhoA in neutrophils. GEF115 was found in the leading edges of polarized mouse neutrophils, whereas PDZ Rho-GEF was found in the uropods of differentiated HL-60 cells. Both Rho-GEFs were believed to mediate pertussis toxin-resistant activation of RhoA in these cells. However, a significant portion of RhoA activity in leukocytes are pertussis toxin-sensitive, which is presumably regulated by the α and/or βγ subunits from the G_i proteins. The signaling mechanism for this pertussis toxin-sensitive RhoA regulation by chemokine receptors remains largely elusive.Molecular cloning and genomic sequencing have identified more than 70 Rho-GEFs in mammals (16–20). Many of these Rho-GEFs have been shown to activate RhoA in in vitro and overexpression assays (16–20). However, it is not known if any of them regulate RhoA in vivo, we have found that PIXα is a specific GEF for Cdcd42 in neutrophils (12) despite its potent activity on Rac in in vitro and overexpression assays (21, 22). Therefore, we used a siRNA-based loss of function screen in an attempt to identify the GEFs that regulate myeloid cell migration and RhoA activity. One of the candidates, Arhgef5, was found to be directly activated by Gβγ to regulate RhoA and has an important role in immature DC migration. In addition, Arhgef5 deficiency attenuated allergic airway inflammation in a mouse model.     

AKAP-Lbc: a molecular scaffold for the integration of cyclic AMP and Rho transduction pathways

A Kinase-anchoring proteins (AKAPs) are a family of functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates. We have recently identified a novel anchoring protein expressed in cardiomyocytes, called AKAP-Lbc, that functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA. Here, we discuss the most recent findings elucidating the molecular mechanisms and the transduction pathways involved in the regulation of the AKAP-Lbc signaling complex inside cells. We could show that AKAP-Lbc is regulated in a bi-directional manner by signals that activate or deactivate its Rho-GEF activity. Activation of AKAP-Lbc occurs in response to agonists that stimulate G proteins coupled receptors linked to the heterotrimeric G protein G12, whereas inactivation occurs through mechanisms that require phosphorylation of AKAP-Lbc by anchored PKA and subsequent recruitment of the regulatory protein 14-3-3. Interestingly, we could demonstrate that AKAP-Lbc can form homo-oligomers inside cells and that 14-3-3 can inhibit the Rho-GEF activity of AKAP-Lbc only when the anchoring protein adopts an oligomeric conformation. These findings reveal the molecular architecture of the AKAP-Lbc transduction complex and provide a mechanistic explanation of how upstream signaling pathways can be integrated within the AKAP-Lbc transduction complex to precisely modulate the activation of Rho.     

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.     

Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity

The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

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.     

Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc

AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.     

Involvement of protein kinase C and rho GTPase in the nuclear signalling pathway by transforming growth factor-beta1 in rat-2 fibroblast cells

The transforming growth factor (TGF)-beta signal-transduction cascade from the cell membrane to the nuclear target is poorly characterised. Here we report that treatment with TGF-beta1 induces the levels of endogenous c-fos mRNA in Rat-2 fibroblast cells. In addition, by transient transfection analysis, TGF-beta1 was shown to stimulate c-fos serum response element (SRE)-driven reporter gene activity in a dose- and time-dependent manner, suggesting that SRE is one of the nuclear targets of TGF-beta1. To understand the signalling cascade by which TGF-beta1 mediates the transactivation of c-fos SRE, cells were either pre-treated with various inhibitors or co-transfected with expression plasmids encoding inhibitory proteins for Rho GTPase together with the SRE-luciferase reporter gene. Our results showed that an inhibition of protein kinase C (PKC) or RhoA selectively repressed the stimulation of c-fos SRE by TGF-beta1, implying the possible roles of PKC and RhoA GTPase in TGF-beta1-induced signalling to c-fos SRE.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.