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Metastasis to vital organs remains the leading cause of cancer-related deaths, emphasizing an urgent need for actionable targets in advanced-stage cancer. The role of mitochondrial Rho GTPase 2 (MIRO2) in prostate cancer growth was recently reported; however, whether MIRO2 is important for additional steps in the metastatic cascade is unknown. Here, we show that knockdown of MIRO2 ubiquitously reduces tumor cell invasion in vitro and suppresses metastatic burden in prostate and breast cancer mouse models. Mechanistically, depletion of MIRO2's binding partner—unconventional myosin 9B (MYO9B)—reduces tumor cell invasion and phenocopies MIRO2 depletion, which in turn results in increased active RhoA. Furthermore, dual ablation of MIRO2 and RhoA fully rescues tumor cell invasion, and MIRO2 is required for MYO9B-driven invasion. Taken together, we show that MIRO2 supports invasion and metastasis through cooperation with MYO9B, underscoring a potential targetable pathway for patients with advanced disease. [Display omitted] • MIRO2 supports the metastatic potential of breast and prostate epithelial cancer cells • MIRO2 forms a complex with MYO9B and RhoA and suppresses RhoA activity • MYO9B-dependent cell invasion requires its GAP activity and MIRO2 expression • RhoA knockdown restores cell invasion of MIRO2-depleted cancer cells Boulton et al. report that MIRO2 is required for breast and prostate cancer invasion and metastasis. This role is mediated via interaction with MYO9B and repression of RhoA activation. This signaling may be relevant in patients, as higher MIRO2 expression is associated with downregulation of RhoA-specific genes. [ABSTRACT FROM AUTHOR]

Rac-GEFs operate in a nonredundant manner as downstream effectors of receptor tyrosine kinases to promote ruffle formation, indicative of unique modes of regulation and targeting. Current research is shedding light on the intricate signaling paradigms shaping spatiotemporal activation of the small GTPase Rac during the generation of actin-rich membrane protrusions. [ABSTRACT FROM AUTHOR]

The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs). [ABSTRACT FROM AUTHOR]

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD. • Enhanced temperature causes ectopic activation of Rho GTPases in MKD fibroblasts. • Cell cycle-related genes are downregulated in MKD fibroblasts. • Actin cytoskeleton-related genes are upregulated in MKD fibroblasts. • MKD affects cell cycle progression and actin cytoskeleton dynamics. • The expression of glycolytic genes is increased in MKD fibroblasts. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin complex 1 (mTORC1) is a highly evolutionarily conserved serine/threonine kinase that regulates cell growth and metabolism in response to multiple environmental cues, such as nutrients, hormones, energy, and stress. Deregulation of mTORC1 can lead to diseases such as diabetes, obesity, and cancer. A series of small GTPases, including Rag, Ras homolog enriched in brain (Rheb), adenosine diphosphate ribosylation factor 1 (Arf1), Ras-related protein Ral-A, Ras homolog (Rho), and Rab, are involved in regulating mTORC1 in response to nutrients, and mTORC1 is differentially regulated via these small GTPases according to specific conditions. Leucine and arginine sensing are considered to be well-confirmed amino acid-sensing signals, activating mTORC1 via a Rag GTPase-dependent mechanism as well as the Ragulator complex and vacuolar H+-adenosine triphosphatase (v-ATPase). Glutamine promotes mTORC1 activation via Arf1 independently of the Rag GTPase. In this review, we summarize current knowledge regarding the regulation of mTORC1 activity by small GTPases in response to nutrients, focusing on the function of small GTPases in mTORC1 activation and how small GTPases are regulated by nutrients. [ABSTRACT FROM AUTHOR]

Biological complexity is challenging to define, but can be considered through one or more features, including overall genome size, number of genes, morphological features, multicellularity, number of life cycle stages and the ability to adapt to different environments. Euglena gracilis meets several of these criteria, with a large genome of ∼38,000 protein coding genes and a considerable ability to survive under many different conditions, some of which can be described as challenging or harsh. Potential molecular exemplars of complexity tying these aspects together are signalling pathways, including GTPases, kinases and ubiquitylation, which increase the functionality of the gene-encoded proteome manyfold. Each of these examples can modulate both protein activity and gene expression. To address the connection between genome size and complexity I have undertaken a brief, and somewhat qualitative, survey of the small ras-like GTPase superfamily of E. gracilis. Unexpectedly, apart from Rab-GTPases which control intracellular transport and organelle identify, the size of the GTPase cohort is modest, and, for example, has not scaled with gene number when compared to the close relatives, trypanosomatids. I suggest that understanding the functions of this protein family will be vital to uncovering the complexity of E. gracilis biology. [ABSTRACT FROM AUTHOR]

• The small GTPase Rab22a is a key organizer of intracellular membrane transport. • Rab22a activity regulates MHC-I trafficking and stimulates antigen presentation. • Many pathogens hijack Rab22a functions for infection and intracellular survival. • Rab22a is overexpressed in cancer cells favoring tumor progression. Dendritic cells (DCs) trigger CD8 + T cell responses after the internalization of exogenous antigens in a process called cross-presentation. Multiple intracellular transport events within the endocytic and secretory routes take place in order to accomplish this fundamental immunological process. The endomembrane system can be envisioned as a complex network of membrane domains coordinately working in the fusion of organelles, the budding of vesicles and tubules, and modifying the molecular composition of the limiting membranes. In this context of tightly regulated and dynamic endomembrane transport, small GTPases of the Rab family display a pivotal role by organizing membrane microdomains and defining specific identities to the different intracellular compartments. In this review, we synthesize and update the current knowledge about Rab22a, which has been involved in several immune functions. In this way, we analyze the intracellular localization of Rab22a and its important role in the endocytic recycling, including its relevance during MHC-I trafficking, antigen cross-presentation by DCs and the formation of T cell conjugates. We also describe how different pathogenic microorganisms hijack Rab22a functions to achieve efficient infection and intracellular survival strategies. Furthermore, we examine the oncogenic properties of Rab22a and how its expression determines the progression of many tumors. In summary, we highlight the role of Rab22a as a key effector of the intracellular trafficking that could be exploited in future therapies to modulate the immune system. [ABSTRACT FROM AUTHOR]

The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization. [ABSTRACT FROM AUTHOR]

Isoprenoids and prenylated proteins regulate a variety of cellular functions, including neurite growth and synaptic plasticity. Importantly, they are implicated in the pathogenesis of several diseases, including Alzheimer’s disease (AD). Recently, we have shown that two protein prenyltransferases, farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT), have differential effects in a mouse model of AD. Haplodeficiency of either FT or GGT attenuates amyloid-β deposition and neuroinflammation but only reduction in FT rescues cognitive function. The current study aimed to elucidate the potential mechanisms that may account for the lack of cognitive benefit in GGT-haplodeficient mice, despite attenuated neuropathology. The results showed that the magnitude of long-term potentiation (LTP) was markedly suppressed in hippocampal slices from GGT-haplodeficient mice. Consistent with the synaptic dysfunction, there was a significant decrease in cortical spine density and cognitive function in GGT-haplodeficient mice. To further study the neuron-specific effects of GGT deficiency, we generated conditional forebrain neuron-specific GGT-knockout (GGT f/f Cre+) mice using a Cre/LoxP system under the CAMKIIα promoter. We found that both the magnitude of hippocampal LTP and the dendritic spine density of cortical neurons were decreased in GGT f/f Cre+ mice compared with GGT f/f Cre− mice. Immunoblot analyses of cerebral lysate showed a significant reduction in cell membrane-associated (geranylgeranylated) Rac1 and RhoA but not (farnesylated) H-Ras, in GGT f/f Cre+ mice, suggesting that insufficient geranylgeranylation of the Rho family of small GTPases may underlie the detrimental effects of GGT deficiency. These findings reinforce the critical role of GGT in maintaining spine structure and synaptic/cognitive function in development and in the mature brain. [ABSTRACT FROM AUTHOR]

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation. [ABSTRACT FROM AUTHOR]

Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the "other" group of typical protein kinases. This was mostly due to species-specific single gene amplification of "other" kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest. • We investigated conservation and change in protein kinases across Dictyostelia • Most kinases were conserved inclusive of functional domain architecture • Gene expression profiles of kinases and ∼1000 other regulatory proteins were combined • The data set was hierarchically clustered to identify sets of co-regulated genes • The analysis clustered interacting proteins from subsets of known regulatory networks [ABSTRACT FROM AUTHOR]

Tamoxifen is a standard endocrine therapy for estrogen receptor positive breast cancer patients. Despite its success, development of resistance mechanisms is still a serious clinical problem. Deregulation of survival signaling pathways play a key role in tamoxifen resistance, being upregulation of Rac1-PAK1 signaling pathway one of the most important. Here, we report the development of the breast cancer cell model MCF7::C1199 having Rac1 enhanced activity with the aim of evaluating the role of Rac1 in acquired endocrine resistance. These cells not only showed distinctive features of Rac1-regulated process as increased migration and proliferation rates, but also showed that upregulation of Rac1 activity triggered a hormonal-independent and tamoxifen resistant phenotype. We also demonstrated that PAK1 activity increases in response to Tamoxifen, increasing phosphorylation levels of estrogen receptor at Ser305, a key phosphorylation site involved in tamoxifen resistance. Finally, we evaluated the effect of 1A-116, a specific Rac1 inhibitor developed by our group, in tamoxifen-resistant cells. 1A-116 effectively restored tamoxifen anti-proliferative effects, switched off PAK1 activity and decreased estrogen receptor phospho-Ser305 levels. Since combination schemes of novel targeted agents with endocrine therapy could be potential new strategies to restore tamoxifen sensibility, these results show that inhibition of Rac1-PAK1 signaling pathway may provides benefits to revert resistance mechanisms in endocrine therapies. [ABSTRACT FROM AUTHOR]

Synaptic connectivity forms the basis for neuronal communication and the storage of information. Experiences and learning of new abilities can drive remodelling of this connectivity and promotes the formation of spine clusters; dendritic segments with a higher spine density. Spines located within these segments are frequently co-activated, undergo different dynamics than synapses located outside of this dendritic compartment and have, in general, a longer lifetime. Several lines of evidence have shown that chemical synapses located close to each other share or compete for intracellular signalling molecules and structural resources. This sharing and competition directly influences spine dynamics. Spines can grow, shrink, increase or decrease the surface expression of receptors, channels and adhesion molecules or remain stable and unchanged over extended periods of time. Here we summarize recent discoveries and provide a closer look at spine clustering, dendritic segment-specific signalling and potential molecular mechanisms underlying associative and heterosynaptic plasticity. [ABSTRACT FROM AUTHOR]

In this review article, we summarize current knowledge on peroxisome biogenesis/functions and the role that small GTPases may play in these processes. Precise intracellular distribution of cell organelles requires their regulated association to microtubules and the actin cytoskeleton. In this respect, RhoGDP/RhoGTP favor binding of peroxisomes to microtubules and actin filaments. In its GTP-bound form, RhoA activates a regulatory cascade involving Rho kinaseII and non-muscle myosinIIA. Such interactions frequently depend on phosphoinositides (PIs) of which PI4P, PI(4,5)P 2 , and PI(3,5)P 2 were found to be present in the peroxisomal membrane. PIs are pivotal determinants of intracellular signaling and known to regulate a wide range of cellular functions. In many of these functions, small GTPases are implicated. The small GTPase ADP-ribosylation factor 1 (Arf1), for example, is known to stimulate synthesis of PI4P and PI(4,5)P 2 on the Golgi to regulate protein and lipid sorting. In vitro binding assays localized Arf1 and the COPI complex to peroxisomes. In light of the recent discussion of pre-peroxisomal vesicle generation at the ER, peroxisomal Arf1-COPI vesicles may serve retrograde transport of ER-resident components. A mass spectrometric screen localized various Rab proteins to peroxisomes. Overexpression of these proteins in combination with laser-scanning fluorescence microscopy co-localized Rab6, Rab8, Rab10, Rab14, and Rab18 with peroxisomal structures. By analogy to the role these proteins play in other organelle dynamics, we may envisage what the function of these proteins may be in relation to the peroxisomal compartment. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann. [ABSTRACT FROM AUTHOR]

Rho GTPases like RAC1 are localized on the inner side of the outer cell membrane where they act as molecular switches that can trigger signal transduction pathways in response to various extracellular stimuli. Nuclear functions of RAC1 were identified that are related to mitosis, cell cycle arrest and apoptosis. Previously, we showed that RAC1 plays a role in the doxorubicin (Dox)-induced DNA damage response (DDR). In this context it is still unknown whether cytosolic RAC1 modulates the Dox-induced DDR or if a nuclear fraction of RAC1 is involved. Here, we silenced RAC1 in mouse embryonic fibroblasts (MEF) pharmacologically with EHT1864 or by using siRNA against Rac1. Additionally, we transfected MEF with RAC1 mutants (wild-type, dominant-negative, constitutively active) containing a nuclear localization sequence (NLS). Afterwards, we analysed the Dox-induced DDR by evaluation of fluorescent nuclear γH2AX and 53BP1 foci formation, as well as by detection of activated proteins of the DDR by western blot to elucidate the role of nuclear RAC1 in the DDR. Treatment with EHT1864 as well as Rac1 knock-down reduced the Dox-induced DSB-formation to a similar extent. Enhanced nuclear localization of dominant-negative as well as constitutively active RAC1 mimicked these effects. Expression of the RAC1 mutants altered the Dox-induced amount of pP53 and pKAP1 protein. The observed effects were independent of S1981 ATM phosphorylation. We conclude that RAC1 is required for a substantial activation of the Dox-induced DDR and balanced levels of active/inactive RAC1 inside the nucleus are a prerequisite for this response. [Display omitted] • RAC1, but not RAC2 or RAC3 is involved in the doxorubicin-induced DNA damage response (DDR) in mouse embryonic fibroblasts • Active, nuclear Rac1 is a prerequisite for a full-blown doxorubicin-induced DDR • The function of RAC1 in the DDR is dependent on the mechanism of DNA double-strand break induction and independent of ATM [ABSTRACT FROM AUTHOR]

Podocyte foot processes are an important cellular layer of the glomerular barrier that regulates glomerular permeability. Insulin via the protein kinase G type Iα (PKGIα) signaling pathway regulates the balance between contractility and relaxation (permeability) of the podocyte barrier by regulation of the actin cytoskeleton. This mechanism was shown to be disrupted in diabetes. Rho family guanosine-5′-triphosphates (GTPases) are dynamic modulators of the actin cytoskeleton and expressed in cells that form the glomerular filtration barrier. Thus, changes in Rho GTPase activity may affect glomerular permeability to albumin. The present study showed that Rho family GTPases control podocyte migration and permeability. Moreover these processes are regulated by insulin in PKGIα-dependent manner. Modulation of the PKGI-dependent activity of Rac1 and RhoA GTPases with inhibitors or small-interfering RNA impair glomerular permeability to albumin. We also demonstrated this mechanism in obese, insulin-resistant Zucker rats. We propose that PKGIα-Rac1-RhoA crosstalk is necessary in proper organization of the podocyte cytoskeleton and consequently the stabilization of glomerular architecture and regulation of filtration barrier permeability. • PKGIα-Rac1-RhoA crosstalk is involved in organization of the podocyte cytoskeleton. • Insulin via Rho family GTPases controls podocyte migration and permeability. • PKGIα modulates activity of Rac1 and RhoA GTPases in podocytes. [ABSTRACT FROM AUTHOR]

Abstract: The Obg/Era proteins of P-loop GTPase superclass are conserved in various prokaryotic and eukaryotic organisms. Some of these proteins are critical regulators of many aspects of basic cellular processes, including ribosome biogenesis, translation and signal transduction. However, a genome-wide overview of the Obg/EraGTPase genes in plants is not available. Recentlystudies on comprehensive genomic analyses of Arabidopsis thaliana identified 11 Obg-Hflx like GTPase genes that are divided into nine families/subfamilies: Archaea-related Drg and Nog1, and Eubacteria-related Obg, EngB, HflX, Era, TrmE, EngA, and EngD. In this study we found that Arabidopsis has 3 (three) Drg homologue proteins, namely AtDrg1-1, AtDrg1-2 and AtDrg1-3. Subcellular localization analysis of Arabidopsis Obg-Hflx homologues revealed that Archaea-derived Drg proteins are mainly targeted to cytoplasm, except Drg1-3 was detected not only in cytoplasm but also in nucleus. Furthermore, based on expression pattern profiling indicates that Drg1-1 and Drg1-2 were expressed constitutively trough plant development. On the other hand, Drg1-3 was detected in dry seed and under stress plant. [Copyright &y& Elsevier]

Small GTPase or Ras superfamily, including Ras, Rho, Rab, Ran and Arf, are fundamental in regulating a wide range of cellular processes such as growth, differentiation, migration and apoptosis. They share structural and functional similarities for binding guanine nucleotides and hydrolyzing GTP. Dysregulations of Ras proteins are involved in the pathophysiology of multiple human diseases, however there is still a stringent need for effective treatments targeting these proteins. For decades, small GTPases were recognized as 'undruggable' targets due to their complex regulatory mechanisms and lack of deep pockets for ligand binding. NMR has been critical in deciphering the structural and dynamic properties of the switch regions that are underpinning molecular switch functions of small GTPases, which pave the way for developing new effective inhibitors. The recent progress of drug or lead molecule development made for small GTPases profoundly delineated how modern NMR techniques reshape the field of drug discovery. In this review, we will summarize the progress of structural and dynamic studies of small GTPases, the NMR techniques developed for structure-based drug screening and their applications in early-stage drug discovery for small GTPases. [ABSTRACT FROM AUTHOR]

Abstract: Cell migration is a key event in physiological processes such as embryonic development, tissue repair, angiogenesis and immune responses. Alteration of the migration program is an important component in multiple pathologies, including chronic inflammation, autoimmunity and tumor metastasis. Understanding of the precise mechanisms at the basis of cellular migration may lead to the identification of novel therapeutic approach for these diseases. Recent evidences show that the interplay between the lipid kinases phosphatidylinositol 3-kinase (PI3Ks) and small GTPases play a critical role in driving cell migration. In this review we will describe the role of these molecules and the interaction between their signal cascades in leukocyte polarization and amoeboid migration. [Copyright &y& Elsevier]

Abstract: Changes in extracellular pH are common events in both pathological conditions and during normal brain function. In organs other than the brain, cells may respond to pH changes by trafficking of acid–base transporters. However, regulation of neuronal acid–base transporters during pH shifts is not understood. The aim of this study was to investigate regulatory mechanisms of the variants of the electrogenic sodium/bicarbonate cotransporter 1, NBCe1-A and NBCe1-B/C, in neurons following changes of extracellular pH. Therefore, primary mouse hippocampal neurons were exposed to extracellular acidosis or alkalosis. We show that acid–base changes regulated trafficking and membrane expression of neuronal NBCe1 but the underlying molecular cues were distinct for individual NBCe1 variants. Following extracellular acidosis NBCe1-A was recruited from intracellular pools to the plasma membrane, followed by increased membrane expression, whereas NBCe1-B/C was retrieved from the membrane. Extracellular alkalosis had no impact on NBCe1-A, but caused translocation of NBCe1-B/C toward the dendrites. We also show that acidosis-induced NBCe1-A, but not NBCe1-B/C, trafficking is mediated by Rab8a. Rab8a is expressed in hippocampal neurons, co-localizes, and interacts with NBCe1-A. Loss-of-function of Rab8a using specific siRNA prevented acidosis-induced redistribution of NBCe1-A. These data propose opposite recruitment pattern for NBCe1 variants in neurons following extracellular acid–base changes, implicating distinct physiological functions of individual NBCe1 variants, and introduce Rab8a as a novel molecular determinant and crucial mediator of acidosis-induced NBCe1 trafficking in neurons. [Copyright &y& Elsevier]

Abstract: To understand the role of small GTPases in response to abiotic stress, we isolated a gene encoding a small GTPase, designated MfARL1, from a subtracted cDNA library in Medicago falcata, a native legume species in semi-arid grassland in northern China. The function of MfARL1 in response to salt stress was studied by expressing MfARL1 in Arabidopsis. Wild-type (WT) and transgenic plants constitutively expressing MfARL1 showed comparable phenotype when grown under control conditions. Germination of seeds expressing MfARL1 was less suppressed by salt stress than that of WT seeds. Transgenic seedlings had higher survival rate than WT seedlings under salt stress, suggesting that expression of MfARL1 confers tolerance to salt stress. The physiological and molecular mechanisms underlying these phenomena were elucidated. Salt stress led to a significant decrease in chlorophyll contents in WT plants, but not in transgenic plants. Transgenic plants accumulated less amounts of H2O2 and malondialdehyde than their WT counterparts under salt stress, which can be accounted for by the higher catalase activities, lower activities of superoxide dismutase, and peroxidase in transgenic plants than in WT plants. Transgenic plants displayed lower Na+/K+ ratio due to less accumulation of Na+ than wild-type under salt stress conditions. The lower Na+/K+ ratio may result from less accumulation of Na+ due to reduced expression of AtHKT1 that encodes Na+ transporter in transgenic plants under salt stress. These findings demonstrate that MfARL1 encodes a novel stress-responsive small GTPase that is involved in tolerance to salt stress. [Copyright &y& Elsevier]

Abstract: There is growing evidence that aminobisphosphonates like ibandronate show anticancer activity by an unknown mechanism. Biochemically, they prevent posttranslational isoprenylation of small GTPases, thus inhibiting their activity. In tumor cells, activated RAS-GTPase, the founding member of the gene family, down-regulates the expression of the pro-apoptotic gene FAS via epigenetic DNA-methylation by DNMT1. We compared ibandronate treatment in neoplastic human U-2 osteosarcoma and in mouse CCL-51 breast cancer cells as well as in the immortalized non-neoplastic MC3T3-E1 osteoblastic cells. Ibandronate attenuated cell proliferation in all cell lines tested. In the neoplastic cells we found up-regulation of caspases suggesting apoptosis. Further we found stimulation of FAS-expression as a result of epigenetic DNA demethylation that was due to down-regulation of DNMT1, which was rescued by re-isoprenylation by both geranylgeranyl-pyrophosphate and farnesylpyrophosphate. In contrast, ibandronate did not affect FAS and DNMT1 expression in MC3T3-E1 non-neoplastic cells. Data suggest that bisphosphonates via modulation of the activity of small-GTPases induce apoptosis in neoplastic cells by DNA-CpG-demethylation and stimulation of FAS-expression. In conclusion the shown epigenetic mechanism underlying the anti-neoplastic activity of farnesyl-transferase-inhibition, also explains the clinical success of other drugs, which target this pathway. [Copyright &y& Elsevier]

Abstract: Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. [Copyright &y& Elsevier]

Abstract: Estrogens have been shown to exert powerful effects on cognitive behaviors mediated by several areas of the brain including the cortex. Remodeling of spiny synapses is a key step in the rewiring of neuronal circuitry thought to underlie the processing and storage of information in the forebrain. Whereas estrogen has been shown to regulate synapse structure and function, we are only just starting to understand the molecular and cellular underpinnings of how estrogens can modulate neuronal circuits. Here I will review recent molecular and cellular work that offers a potential mechanism of how estrogen may modulate synapse structure and function of cortical neurons. This mechanism allows cortical neurons to respond to activity-dependent stimuli with greater efficacy in a cellular model termed “Two-Step Wiring Plasticity”. This novel form of spine plasticity thus provides insight into how estrogens may modulate the rewiring of neuronal circuits, underlying its ability to influencing cortically based behaviors. This article is part of a Special Issue entitled ‘Neurosteroids’. [Copyright &y& Elsevier]

To date, most studies of Rho GTPase regulation have focused on the classic GTPase cycle – GTP binding and hydrolysis – controlled by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and GDP-dissociation inhibitors (GDIs). Recent investigations have unveiled important additional regulatory mechanisms: microRNA (miRNA) regulating post-transcriptional processing of Rho GTPase-encoding mRNAs; palmitoylation and nuclear targeting affecting intracellular distribution; post-translational phosphorylation, transglutamination and AMPylation impacting Rho GTPase signaling; and ubiquitination controlling Rho GTPase protein stability and turnover. These modes of regulation add to the complexity of the Rho GTPase signaling network and allow precise spatiotemporal control of individual Rho GTPases. This review discusses these ‘unconventional’ modes of regulation and their contribution to cellular function, focusing on post-transcriptional and post-translational events beyond the classic GTPase cycle regulatory model. [ABSTRACT FROM AUTHOR]

Background & Aims: Saturated free fatty acid (SFA)-stimulated c-Jun NH2-terminal kinase (JNK) activation is associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the effects of SFA are incompletely understood. The goal of this study was to determine the molecular mechanisms by which SFA induce JNK activation in hepatocytes. Methods: We used siRNA-mediated knockdown in Hepa1c1c7 and AML12 cell lines, as well as primary mouse hepatocytes for these studies. Results: The current model for JNK activation by SFA involves endoplasmic reticulum (ER) stress, which induces JNK activation through an inositol requiring enzyme 1 (IRE1α) Apoptosis Regulating Kinase 1 (ASK1)-dependent mechanism. Here, we find that SFA-induced JNK activation is not inhibited in the absence of IRE1α and ASK1. Instead we show that activation of the small GTP-binding proteins Cdc42 and Rac1 is required for SFA-stimulated MLK3-dependent activation of JNK in hepatocytes. In addition, we demonstrate that SFA-induced cell death in hepatocytes is independent of IRE1α, but dependent on Cdc42, Rac1, and MLK3. Conclusions: Our results demonstrate that Cdc42 and Rac1, rather than ER stress, are important components of a SFA-stimulated signaling pathway that regulates MLK3-dependent activation of JNK in hepatocytes. [ABSTRACT FROM AUTHOR]

Abstract: Lovastatin (Lov), bisphosphonates (BP) and metformin (Met) are widely used drugs, having in common that they interfere with the mevalonate pathway (MP). The MP generates isoprene moieties required for the function of regulatory GTPases controlling cell proliferation and survival. Here, we addressed the question whether MP inhibitors interfere with the anti-tumor efficacy of anticancer drugs. We comparatively analyzed the effect of equitoxic doses of Lov, BP and Met on cell viability, cell cycle progression, apoptosis and DNA damage response (DDR) of human osteo- and fibrosarcoma cells exposed to doxorubicin or cisplatin. We found that Lov, BP and Met modulated the anticancer drug sensitivity of sarcoma cells in an agent-, dose and time-dependent fashion. Mostly, the MP inhibitors increased the cytotoxicity of the anticancer drugs in an additive manner. MP modulators differed from each other regarding their impact on anticancer drug-induced DNA damage response as measured by the phosphorylation status of SAPK/JNK, Chk-1 and H2AX as well as p53 protein level. In this regard, lovastatin and metformin turned out as the most effective inhibitory drugs. The data show that MP inhibitors can affect the anti-tumor efficacy of anticancer drugs and impact the DDR of human sarcoma cells. [Copyright &y& Elsevier]

Abstract: Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor in adults. This cancer has an infiltrative nature and the median survival of patients is about one year. Vasoactive intestinal peptide (VIP) belongs to a structurally related family of polypeptides and is a major regulatory factor in the central and peripheral nervous systems. VIP regulates proliferation of astrocytes and of numerous cancer cell lines and modulates migration in prostatic and colonic cancer cell lines. Little is known about the involvement of VIP and its receptors (VIP-receptor system) in proliferation or migration of GBM cells. The effects of VIP, PACAP and of synthetic VIP antagonists were tested in two human GBM cell lines, M059K and M059J, established from two different parts of a single tumor. In these cells, the data revealed that the VIP-receptor system did not affect proliferation but controlled cell migration. Indeed, in M059K cells which express components of the VIP receptor system, the VIP receptor antagonists and a PACAP antibody enhanced migration. The VIP receptor antagonists increased generation of typical migration-associated processes: filopodia and lamellipodia, and activation of Rac1 and Cdc42 GTPases. Reciprocally, in M059J cells which poorly express the VIP-receptor system, treatments with the agonists VIP and PACAP resulted in decreased cell migration. Furthermore, the peptides appeared to act through a subclass of binding sites displaying an uncommon very high affinity for these ligands. Taken together, these observations suggest that components of the VIP-receptor system negatively regulate cell migration, thus showing potential anti-oncogenic properties. [Copyright &y& Elsevier]

Abstract: The mevalonate pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased but that dolichyl phosphate and ubiquinone are elevated in brains of Alzheimer′s disease (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post-translational modification of certain proteins which function as molecular switches in numerous signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction. [Copyright &y& Elsevier]

Abstract: Studies indicate that both alterations in leukocyte and endothelial cell adhesion molecules and the renin angiotensin system are involved in the pathogenesis of atherosclerosis processes in human hypertension. The present work was undertaken to investigate whether angiotensin II (Ang II) regulates the expression of CD62L on human neutrophils. Human neutrophils were stimulated with Ang II in the presence of various AT1-receptor antagonists and protein kinase inhibitors, and CD62L cell surface expression was detected by flow cytometry. We report for the first time that Ang II down-regulated CD62L from the surface of human neutrophils, a process which was independent of neutrophil adhesion to endothelium since neutrophils were still able to adhere to human umbilical vein endothelial cells even under doses that almost completely release CD62L from the cell surface. This process occurred through pathways involving AT1 receptors, extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and calcineurin, ruling out a role for p38 MAPK and small GTPases in the process. [Copyright &y& Elsevier]

Abstract: Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells. [Copyright &y& Elsevier]

Abstract: Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration. [Copyright &y& Elsevier]

Abstract: Studies on a variety of cell lines have shown that p120-catenin can directly regulate the stability of E-cadherin complexes and control the activity of small GTPases to influence cell adhesion. Despite this data, clinical studies of human solid tumors have not been reported to investigate these protein interactions. To explore the correlation between p120-catenin, E-cadherin, and small GTPases in human lung cancer, we examined the expression patterns of p120-catenin, E-cadherin, RhoA, Cdc42, and Rac1, and their prognostic significance in 138 patients with non-small cell lung cancer (NSCLC). While normal bronchial epithelium showed strong membrane expression of p120-catenin and E-cadherin, lung cancer tissues had reduced membrane expression and ectopic cytoplasmic expression of p120-catenin and E-cadherin. Expression of RhoA, Cdc42, and Rac1 was also found to be higher in tumor tissue than in normal lung tissue. A correlation between abnormal p120-catenin, E-cadherin expression, and overexpression of specific small GTPases was also associated with poor differentiation, high TNM stage, and lymph node metastasis in NSCLC patients. We also used an in vitro model to evaluate their expression, and to determine whether protein expression correlated with the invasive capacity of lung cancer cell lines. Consistent with our in vivo data, abnormal expression of p120-catenin and E-cadherin with overexpression of specific small GTPases were significantly associated with the high metastatic capacity of BE1 cells. Based on our results, we conclude that abnormal p120-catenin expression correlates with abnormal E-cadherin expression and specific small GTPase overexpression, which contribute to the malignancy-related to NSCLC. [Copyright &y& Elsevier]

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.Kidney International (2009) 75, 31–40; doi:10.1038/ki.2008.448; published online 10 September 2008 [ABSTRACT FROM AUTHOR]

Abstract: Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/γcnull mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy. [Copyright &y& Elsevier]

Abstract: Meiotic divisions during oogenesis in higher eukaryotes are extremely asymmetric giving rise to one gamete, the oocyte, and two polar bodies. In most species, this asymmetric partitioning relies on the eccentric positioning of meiotic spindles. Recent work performed in mouse and frog oocytes has suggested the involvement of small GTPases, such as Cdc42, Rac and Ran both in the control of spindle organization and positioning. The present review summarizes these findings that shed light on the molecular mechanisms by which small GTPases control asymmetric cell divisions in vertebrate oocytes. [Copyright &y& Elsevier]

Abstract: RAS proteins are small GTPases, which serve as master regulators of a myriad of signaling cascades involved in highly diverse cellular processes. RAS oncogenes have been originally discovered as retroviral oncogenes, and ever since constitutively activating RAS mutations have been identified in human tumors, they are in the focus of intense research. In this review, we summarize the biochemical properties of RAS proteins, trace down the evolution of RAS signaling and present an overview of the spatio-temporal activation of major RAS isoforms. We further discuss RAS effector pathways, their role in normal and transformed cell physiology and summarize ongoing attempts to interfere with aberrant RAS signaling. Finally, we comment on the role of micro RNAs in modulating RAS expression, contribution of RAS to stem cell function and on high-throughput analyses of RAS signaling networks. [Copyright &y& Elsevier]

Abstract: Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the β2 subunit of the Na+/K+-ATPase, as verified according to the recently proposed “β-profiling test” [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+-ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+-activated RBCs, suggesting a possible role for this GTPase in membrane shedding. [Copyright &y& Elsevier]

Abstract: The effects of lipo-chitin oligosaccharide Nod factors (NodNGR[S] from Rhizobium sp. NGR234) on root hair deformation in Vigna unguiculata (L.) Walp. were studied using pharmacological agents to mimic and/or inhibit their action. It was hypothesised that the rearrangement of the cytoskeleton seen during Nod factor induced root hair deformation is modulated by protein kinase C, monomeric G proteins of the Rho superfamily and the location and amount of phosphatidylinositol 3-phosphates (PI3Ps). This hypothesis is supported by the following observations. The protein kinase C activators, 12-deoxyphorbol 13-acetate (DPA) and diacylglycerol kinase inhibitor 1, stimulated root hair deformation to a level similar to that seen with Nod factors or mastoparan, whereas the inhibitor Gö 6976 inhibited root hair deformations induced by NodNGR[S], mastoparan, DPA and diacylglycerol kinase inhibitor 1. The Ras antagonists mevastatin and sulindac sulphide, and the Rho antagonist exoenzyme C3 toxin from Clostridium botulinum all inhibited Nod factor stimulated root hair deformation. Pasteurella multocida toxin activates Rho and stimulated root hair deformation, this stimulation was inhibited by both neomycin and exoenzyme C3 toxin. The PI3 kinase inhibitors, wortmannin and LY-294002 attenuated Nod factor induced root hair deformation. These studies were complemented with actin immunoprecipitations of root hair enriched microsomal membrane preparations from V. unguiculata which pulled down small GTP binding proteins. Root hair deformation is an important early stage in the formation of nitrogen fixing nodules and this study highlights that these processes may depend on signalling cascades involving phospholipids and small GTP binding proteins. [Copyright &y& Elsevier]

Abstract: Rab proteins are small GTPases involved in the regulation of vesicular membrane traffic. Research done in the past years has demonstrated that some of these proteins are under the control of signal transduction pathways. Still, several recent papers point out to a new unexpected role for this family of Ras-related proteins, as potential regulators of intracellular signaling pathways. In particular, several evidence indicate that members of the Rab family of small GTPases, through their effectors, are key molecules participating to the regulation of numerous signal transduction pathways profoundly influencing cell proliferation, cell nutrition, innate immune response, fragmentation of compartments during mitosis and apoptosis. Even more surprisingly, direct involvement of Rab proteins in signaling to the nucleus has been demonstrated. This review will focus on aspects of Rab proteins function connected to signal transduction and, in particular, connections between membrane traffic and other cell pathways will be examined. [Copyright &y& Elsevier]

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab protein function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (Griscelli syndrome), mental retardation, neuropathy (Charcot–Marie–Tooth), kidney disease (tuberous sclerosis), and blindness (choroideremia) arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway. [Copyright &y& Elsevier]

Unlike neonatal axons, mammalian adult axons of the CNS do not regenerate after injury. This developmental loss of regenerative capacity, is correlated with the onset of myelination. Likewise, myelin, or myelin-associated components such as Nogo-A and myelin-associated glycoprotein (MAG) inhibit regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is central to devise strategies to encourage regeneration in adults after injury. Endogenous levels of the cyclic nucleotides cAMP and cGMP have been suggested to determine the neuronal responsiveness to various axonal guidance factors. Elevating cAMP concentrations block Nogo-A or MAG induced inhibition of neurite outgrowth in older neurons, whereas suppressing cAMP levels in young neurons renders them susceptible to Nogo-A and MAG. Interestingly, elevated cAMP levels abrogated the Nogo-A and MAG mediated activation of RhoA and down regulation of Rac1 in adult neurons. In contrast, elevation of cAMP leads to the inactivation of RhoA and prevents activation of downstream effector proteins, while Rac is activated. We therefore conclude that the endogenous neuronal cAMP levels determine the neuronal responsiveness to myelin-associated neurite growth inhibitors by regulating rho GTPase activities. [Copyright &y& Elsevier]

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival. [Display omitted] • Invasive tumor borders and cells proximal to vessels show upregulated Rac1 activity • Rac1 inhibition enhances tumor cell vulnerability during fluid-flow shear stress • Live in vivo imaging of Rac1 activity guides optimal disease progression targeting • Rac1 inhibition reduces intratumoral movement and lung metastasis Floerchinger et al. use spatiotemporal imaging of a Rac1-FRET biosensor mouse to dissect the role of Rac1 in mammary carcinoma metastatic cascade. This image-guided inhibition of Rac1 scheduling at primary sites, during fluid-flow induced shear stress and dissemination vulnerabilities, results in a reduction of disease progression and lung metastasis. [ABSTRACT FROM AUTHOR]

Arf GTPase activating (ArfGAP) proteins are critical regulatory and effector proteins in membrane trafficking pathways. Budding yeast contain two ArfGAP proteins (Gcs1 and Glo3) implicated in COPI coat function at the Golgi, and yeast require Glo3 catalytic function for viability. A new X-ray crystal structure of the Glo3 GAP domain was determined at 2.1 Å resolution using molecular replacement methods. The structure reveals a Cys 4 -family zinc finger motif with an invariant residue (R59) positioned to act as an "arginine finger" during catalysis. Comparisons among eukaryotic GAP domains show a key difference between ArfGAP1 and ArfGAP2/3 family members in the final helix located within the domain. Conservation at both the sequence and structural levels suggest the Glo3 GAP domain interacts with yeast Arf1 switch I and II regions to promote catalysis. Together, the structural data presented here provide additional evidence for placing Glo3 near Arf1 triads within membrane-assembled COPI coats and further support the molecular niche model for COPI coat regulation by ArfGAPs. [ABSTRACT FROM AUTHOR]

The activation of G-protein-coupled receptors (GPCRs) leads to the activation of mTORC2 in cell migration and metabolism. However, the mechanism that links GPCRs to mTORC2 remains unknown. Here, using Dictyostelium cells, we show that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho GTPase and GTP-bound Ras GTPase in directed cell migration. The Rho-Ras hetero-oligomers directly and specifically stimulate mTORC2 activity toward AKT in cells and after biochemical reconstitution using purified proteins in vitro. The Rho-Ras hetero-oligomers do not activate ERK/MAPK, another kinase that functions downstream of GPCRs and Ras. Human KRas4B functionally replace Dictyostelium Ras in mTORC2 activation. In contrast to GDP-Rho, GTP-Rho antagonizes mTORC2-AKT signaling by inhibiting the oligomerization of GDP-Rho with GTP-Ras. These data reveal that GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step that controls mTORC2-AKT signaling. • Chemoattractant induces formation of Rho-Ras hetero-oligomers in migrating cells • Rho and Ras interact via a specific pair of charged residues in their α helices • Rho-Ras hetero-oligomers stimulate mTORC2 activity toward AKT • Rho-Ras hetero-oligomers are inhibited at the rear of migrating cells Senoo et al. report that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho and GTP-bound Ras in cell migration. Rho-Ras hetero-oligomers directly stimulate mTORC2 activity toward AKT in cells and in vitro. Therefore, GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step in mTORC2-AKT signaling. [ABSTRACT FROM AUTHOR]

Statins are a group of medications that reduce cholesterol synthesis by inhibiting the activity of HMG-CoA reductase, a key enzyme in the mevalonate pathway. The clinical use of statins to lower excess cholesterol levels has revolutionized the cardiovascular field and increased the survival of millions, but some patients have adverse side effects. A growing body of data suggests that some of the beneficial and adverse effects of statins, including their anti-inflammatory, anti-tumorigenic, and myopathic activities, are cholesterol-independent. However, the underlying mechanisms for these effects of statins are not well defined. Because Caenorhabditis elegans (C. elegans) lacks the cholesterol synthesis branch of the mevalonate pathway, this organism is a powerful system to unveil the cholesterol-independent effects of statins. We used genetic and biochemical approaches in C. elegans and cultured macrophage-derived murine cells to study the cellular response to statins. We found that statins activate a conserved p38-MAPK (p38) cascade and that the protein geranylgeranylation branch of the mevalonate pathway links the effect of statins to the activation of this p38 pathway. We propose that the blockade of geranylgeranylation impairs the function of specific small GTPases we identified as upstream regulators of the p38 pathway. Statin-mediated p38 activation in C. elegans results in the regulation of programs of innate immunity, stress, and metabolism. In agreement with this regulation, knockout of the p38 pathway results in the hypersensitivity of C. elegans to statins. Treating cultured mammalian cells with clinical doses of statins results in the activation of the same p38 pathway, which upregulates the COX-2 protein, a major regulator of innate immunity in mammals. Statins activate an evolutionarily conserved p38 pathway to regulate metabolism and innate immunity. Our results highlight the cytoprotective role of p38 activation under statin treatment in vivo and propose that this activation underlies many of the critical cholesterol-independent effects of statins. • Statins activate a conserved stress-related p38-MAPK in C. elegans and mammalian cells. • The blockadge of geranylgeranylation of small GTPases is a suggested mechanism for p38 activation. • p38 activation can protect C. elegans from the deleterious effects of statins. • Clinical concentrations of statins activate p38 to alter innate immunity programs in mammals. [ABSTRACT FROM AUTHOR]

Summary Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a "regulatory arm" by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse. Highlights • LCK immune synapse focusing requires its strong interaction with ciliary UNC119A • ARL3Q71L results in unfocused LCK localization and increased T cell stimulation • The ciliary ARL3-GEF ARL13B localizes to the immune synapse • UNC119A regulatory arm interacts with LCK kinase domain in a phosphoregulated manner Signaling proteins are focused at the immune synapse, where T cells form an interface with target cells. Stephen, ElMaghloob, and McIlwraith et al. demonstrate that T cells repurpose a mechanism used by the cilia, an organelle that T cells lack, to focus the signaling protein LCK at the immune synapse. [ABSTRACT FROM AUTHOR]