Rho Guanine Nucleotide Dissociation Inhibitor Research Articles

Abstract 1494: Differential Regulation of Rho Guanine Nucleotide Dissociation Inhibitors in Pancreatic Beta Cells Under Metabolic Stress

Abstract 1494: Differential Regulation of Rho Guanine Nucleotide Dissociation Inhibitors in Pancreatic Beta Cells Under Metabolic Stress

RhoGDI1 interacts with PHLDA2, suppresses the proliferation, migration, and invasion of trophoblast cells, and participates in the pathogenesis of preeclampsia.

Preeclampsia (PE) is a pregnancy-associated disease, which is the major cause of mortality on maternity and perinatal infants. It is hypothesized that PE is a consequence of the dysfunction of the trophoblast cells. Pleckstrin homology-like domain, family A, member 2 (PHLDA2) was shown to inhibit the proliferation, migration, and invasion of trophoblast cells in our previous studies. However, the mechanism by which PHLDA2 affects trophoblast cell function has not been clarified. In the current study, co-immunoprecipitation (Co-IP) with mass spectroscopy analysis was used to explore the proteins that interacted with PHLDA2. A total of 291 candidate proteins were found to be associated with PHLDA2. The interaction between PHLDA2 and Rho guanine nucleotide dissociation inhibitor (RhoGDI) 1 was identified by Co-IP and immunofluorescence staining. Western blot analysis indicated that overexpression of PHLDA2 resulted in upregulation of the RhoGDI1 protein levels, which were stabilized in the presence of cycloheximide. Similarly, overexpression of RhoGDI1 promoted PHLDA2 expression and its stability. Furthermore, pull-down and Co-IP results indicated that PHLDA2 repressed the activity of Rho guanosine triphosphate hydrolase family proteins by regulating RhoGDI1 expression. In addition, RhoGDI1 expression was upregulated in the placental tissues of patients with PE. The effects of the suppression of PHLDA2 expression on proliferation, migration, and invasion of trophoblast cells were partly abrogated following knockdown of RhoGDI1. Taken together, the data indicated that RhoGDI1 mediated regulation of PHLDA2 on the biological behavior of trophoblast cells and may participate in the pathophysiology of PE.

Cannabinoid receptor interacting protein 1a interacts with myristoylated Gαi N terminus via a unique gapped β-barrel structure

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid–binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N terminus. However, CRIP1a could not bind the nonmyristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.

2-O-Octadecylascorbic acid represses RhoGDIβ expression and ameliorates DNA damage-induced abnormal spindle orientations.

The appropriate regulation of spindle orientation maintains proper tissue homeostasis and avoids aberrant tissue repair or regeneration. Spindle misorientation due to imbalance or improper functioning leads to a loss of tissue integrity and aberrant growth, such as tissue loss or overgrowth. Pharmacological manipulation to prevent spindle misorientation will enable a better understanding of how spindle orientation is involved in physiological and pathological conditions and will provide therapeutic possibilities to treat patients associated with abnormal tissue function caused by spindle misorientation. N-terminal-deleted Rho guanine nucleotide dissociation inhibitor β (RhoGDIβ/RhoGDI2/LyGDI) produced by caspase-3 activation perturbs spindle orientation in surviving cells following exposure to either ionizing radiation or UVC. Thus, presumably, RhoGDIβ cleaved by caspase-3 activation acts as a determinant of radiation-induced spindle misorientation that promote aberrant tissue repair due to deregulation of directional organization of cell population and therefore becomes a potential target of drugs to prevent such response. The objective of this study was to screen and identify chemicals that suppress RhoGDIβ expression. We focused our attention on ascorbic acid (AA) derivatives because of their impact on the maintenance of skin tissue homeostasis. Here, we screened for AA derivatives that suppress RhoGDIβ expression in HeLa cells and identified a lipophilic derivative, 2-O-octadecylascorbic acid (2-OctadecylAA), as a novel RhoGDIβ inhibitor that ameliorated ionizing radiation-induced abnormal spindle orientations. Among all examined AA derivatives, which were also antioxidative, the inhibition activity was specific to 2-OctadecylAA. Therefore, this activity was not due to simple antioxidant properties. 2-OctadecylAA was previously shown to prevent hepatocellular carcinoma development. Our findings suggest that the anticarcinogenic effects of 2-OctadecylAA are partly due to RhoGDIβ inhibition mechanisms by which spindle orientation perturbations are attenuated. Thus, the molecular targeting features of RhoGDIβ warrant its further development for the treatment or control of spindle orientation abnormalities that affect epithelial homeostasis.

Cullin 3/KCTD5 Promotes the Ubiqutination of Rho Guanine Nucleotide Dissociation Inhibitor 1 and Regulates Its Stability.

Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progression. Here, we show that RhoGDI1 interacts with Cullin 3 (CUL3), a scaffold protein for E3 ubiquitin ligase complexes. Ectopic expression of CUL3 increases the ubiquitination of RhoGDI1. Furthermore, potassium channel tetramerization domain containing 5 (KCTD5) also binds to RhoGDI1 and increases its interaction with CUL3. Ectopic expression of KCTD5 increases the ubiquitination of RhoGDI1, whereas its knockdown by RNA interference has the opposite effect. Depletion of KCTD5 or expression of dominant-negative CUL3 (DN-CUL3) enhances the stability of RhoGDI1. Our findings reveal a previously unknown mechanism for controlling RhoGDI1 degradation that involves a CUL3/KCTD5 ubiquitin ligase complex.

Protein phosphatase 1B dephosphorylates Rho guanine nucleotide dissociation inhibitor 1 and suppresses cancer cell migration and invasion

Rho GTPases control a wide range of cellular processes, and their deregulation is associated with promotion of an aggressive and metastatic tumor phenotype in human cancers. Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays a key role in regulating the activity of Rho GTPases. However, the underlying mechanisms are still unclear. In this study, we show that protein phosphatase 1B (PPM1B) interacts with RhoGDI1 and functions as its phosphatase. Ectopic expression of PPM1B results in dephosphorylation of RhoGDI1 and, thereby, abates the activation of RhoA, Rac1 and CDC42 by epidermal growth factor (EGF). PPM1B overexpression in Hs578T and SKBR3 human breast cancer cells decreases their motility and invasiveness in vitro and cancer metastasis in vivo. In contrast, knockdown of PPM1B in MCF-7 and MDA-MB-468 human breast cancer cells that express endogenous PPM1B enhances EGF-induced RhoGDI1 phosphorylation, activation of Rho GTPases, and cancer cell migration and invasion. Knockdown of RhoA or Rac1 by siRNA reverses the enhanced cell migration seen after PP1MB depletion. Collectively, these results indicate that PPM1B negatively regulates cancer cell motility and invasiveness through dephosphorylating RhoGDI1.

EphrinB1 promotes cancer cell migration and invasion through the interaction with RhoGDI1

Eph receptors and their corresponding ephrin ligands have been associated with regulating cell–cell adhesion and motility, and thus have a critical role in various biological processes including tissue morphogenesis and homeostasis, as well as pathogenesis of several diseases. Aberrant regulation of Eph/ephrin signaling pathways is implicated in tumor progression of various human cancers. Here, we show that a Rho family GTPase regulator, Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the extracellular domain of the cognate EphB2 receptor. Deletion mutagenesis revealed that amino acids 327–334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc) induces RhoA activation and enhances the motility as well as invasiveness of wild-type ephrinB1-expressing cells. These Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (Δ327–334) that is ineffective at interacting with RhoGDI1. Furthermore, ephrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces motility and invasiveness of the SW480 and Hs578T human cancer cell lines. Our study connects the interaction between RhoGDI1 and ephrinB1 to the promotion of cancer cell behavior associated with tumor progression. This interaction may represent a therapeutic target in cancers that express ephrinB1.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits

BackgroundThe delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. MethodsProtein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. ResultsWe identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. ConclusionsWeak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. General InterestsBinders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

Structural and Mechanistic Insights into the Regulation of the Fundamental Rho Regulator RhoGDIα by Lysine Acetylation

Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor α (RhoGDIα). RhoGDIα has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDIα is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDIα function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDIα acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDIα is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDIα-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.

Androgen receptor promotes tamoxifen agonist activity by activation of EGFR in ERα-positive breast cancer.

Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) α-positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI) α, a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDIα and seek to determine AR's contribution to resistance. We engineered ERα-positive cell lines with stable knockdown (KD) of Rho GDIα (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Tam-resistant tumors and cell lines with low Rho GDIα levels exhibited upregulated AR expression. Microarray of Rho GDIα KD cells indicated that activation of EGFR and ERα was associated with Tam treatment. When AR levels were elevated, interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ERα. Tam exhibited agonist activity in AR overexpressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ERα-positive cells with low expression of Rho GDIα.

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 induces beta-2 adrenergic receptor desensitization in β2AR desensitization mice model.

It is well-known that chronic administration of β2AR agonists can induce β2AR desensitization. Our previous study showed that Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) overexpression induced beta-2 adrenergic receptor (β2AR) desensitization in airway smooth muscle cells. The purpose of this study was to further study the function of RhoGDI2 in β2AR desensitization by β2AR desensitization mouse model. Studies were performed using a β2AR desensitization mice model induced by salbutamol. The mice were randomly divided into five groups (n=45): RhoGDI2 overexpression group (n=10); RhoGDI2 siRNA group (n=10); empty viral vector group (n=10); experimental control group (n=10); blank control group-without any drug treatment (n=5). The first four groups were used the same methods and the same dose to establish β2AR desensitization mice model by salbutamol. The first three groups that salbutamol-treated were used for intratracheal delivery of lentiviral vectors. Airway hyperreactivity was measured through a whole-body plethysmograph system. RhoGDI2, β2AR, GRK2 mRNA and protein expression levels were then detected by RT-PCR and western blot analyses in fresh lung tissues. As well as the activity of GRK was assessed by light-dependent phosphorylation of rhodopsin. We successfully constructed β2AR desensitization mouse model. As expected, airway responsiveness after inhaling acetylcholine chloride (Ach) was markedly increased in the RhoGDI2 overexpression group compared to experimental control group and blank control group when concentrations of Ach was 45 mg/mL (all P<0.05), while, it was markedly decreased in the RhoGDI2 siRNA group compared to experimental control group (P<0.05). RhoGDI2, GRK2 expressions and GRK enzymatic activity were significantly increased in RhoGDI2 overexpression group compared to experimental control group and blank control group (all P<0.05). RhoGDI2, GRK2 expressions and GRK enzymatic activity were significantly decreased in RhoGDI2 siRNA group compared to experimental control group and blank control group (all P<0.05). Conversely, β2AR expression were significantly lower in RhoGDI2 overexpression group compared to experimental control group and blank control group (all P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. To sum up, our present studies found that RhoGDI2 might induce β2AR desensitization and GRK2 might take part in RhoGDI2-mediated β2AR desensitization.

TROY Interacts with Rho Guanine Nucleotide Dissociation Inhibitor α (RhoGDIα) to Mediate Nogo-induced Inhibition of Neurite Outgrowth

TROY can functionally substitute p75 to comprise the Nogo receptor complex, which transduces the inhibitory signal of myelin-associated inhibitory factors on axon regeneration following CNS injury. The inhibition of neurite extension relies on TROY-dependent RhoA activation, but how TROY activates RhoA remains unclear. Here, we firstly identified Rho guanine nucleotide dissociation inhibitor α (RhoGDIα) as a binding partner of TROY using GST pull-down combined with two-dimensional gel electrophoresis and mass spectra analysis. The interaction was further confirmed by coimmunoprecipitation in vitro and in vivo. Deletion mutagenesis revealed that two regions of the TROY intracellular domain (amino acids 234-256 and 321-350) were essential for the interaction with RhoGDIα. Secondly, TROY and RhoGDIα were coexpressed in postnatal dorsal root ganglion neurons, cortex neurons, and cerebellar granule neurons (CGNs). Thirdly, TROY/RhoGDIα association was potentiated by Nogo-66 and was independent of p75/RhoGDIα interaction. Fourthly, TROY/RhoGDIα interaction was still able to activate RhoA when p75 was deficient. Furthermore, RhoA activation was decreased dramatically when TROY was knocked down in p75-deficient CGNs cells. Finally, RhoGDIα overexpression abolished RhoA activation and following neurite outgrowth inhibition by Nogo-66 in both wild-type and p75-deficient CGNs. These results showed that the association of RhoGDIα with TROY contributed to TROY-dependent RhoA activation and neurite outgrowth inhibition after Nogo-66 stimulation.

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells.

Beta-2 adrenergic receptor (β2AR) downregulation is critical to asthma rescue therapy; however, tolerance, also known as β2AR or bronchodilator desensitization, mechanisms potentially resulting in life-threatening rescue treatment failure remain poorly understood. Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) gene from ASMCs was amplified by RT-PCR, and RhoGDI2 gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI2 expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI2, β2AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β2AR was observed by radioligand receptor binding assay in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. RhoGDI2 vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI2, GEF and G-protein-coupled receptor kinase 2 (GRK2) expressions significantly increased in the RhoGDI2 overexpression group compared to control and negative control groups (all P<0.05). Conversely, β2AR and GAP expressions were significantly lower in the RhoGDI2 overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. Control and negative control groups exhibiting β2AR density more than 2-fold higher than that observed in the RhoGDI2 overexpression group. RhoGDI2 reduces β2AR density, potentially by reducing β2AR and GAP expressions and increase GEF and GRK2 expressions. Thus, RhoGDI2 is central in cellular β2AR desensitization, though this full mechanism and intermediates merit further investigation.

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

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.

Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression: a proteomic approach

Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study, we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase in FAK (focal adhesion kinase) phosphorylation. In order to further shed light in the process of myoblast-induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected by mechanical stimulation including galectin-1, annexin III and RhoGDI (Rho guanine-nucleotide-dissociation inhibitor). In this study, we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of galectin-1 in mechanically stimulated myoblasts. A potential involvement of galectin-1 in myoblast differentiation is presented.

Proteomic Analysis of Proteins Secreted by HepG2 Cells Treated with Butyl Benzyl Phthalate

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 μM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4–7 and 6–9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization–mass spectrometry–mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1

At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques.