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Simple Summary: The protein arginine N-methyltransferase 5 (PRMT5) has been identified as a promising therapeutic target in various cancers. However, its role in hepatocellular carcinoma (HCC) development has not yet been investigated. This study aims to understand PRMT5′s impact on overall survival, signaling pathways, and downstream gene expression using in silico public databases and our in-house NGS data. Our results revealed an increase in PRMT5 expression in HCC compared to normal liver tissue, and this elevated expression was associated with poorer patient outcomes. Analysis of promoter CpG islands and methylation status suggested potential epigenetic mechanisms driving PRMT5 overexpression in HCC. Pathway analyses found a link between PRMT5 expression and the HIF1α pathway, with Ras-related nuclear protein (RAN) identified as a potential target of PRMT5 in HCC. Protein arginine N-methyltransferase 5 (PRMT5) has been identified as a potential therapeutic target for various cancer types. However, its role in regulating the hepatocellular carcinoma (HCC) transcriptome remains poorly understood. In this study, publicly available databases were employed to investigate PRMT5 expression, its correlation with overall survival, targeted pathways, and genes of interest in HCC. Additionally, we utilized in-house generated NGS data to explore PRMT5 expression in dysplastic nodules compared to hepatocellular carcinoma. Our findings revealed that PRMT5 is significantly overexpressed in HCC compared to normal liver, and elevated expression correlates with poor overall survival. To gain insights into the mechanism driving PRMT5 overexpression in HCC, we analyzed promoter CpG islands and methylation status in HCC compared to normal tissues. Pathway analysis of PRMT5 knockdown in the HCC cells revealed a connection between PRMT5 expression and genes related to the HIF1α pathway. Additionally, by filtering PRMT5-correlated genes within the HIF1α pathway and selecting up/downregulated genes in HCC patients, we identified Ras-related nuclear protein (RAN) as a target associated with overall survival. For the first time, we report that PRMT5 is implicated in the regulation of HIF1A and RAN genes, suggesting the potential prognostic utility of PRMT5 in HCC. [ABSTRACT FROM AUTHOR]

Protein arginine N-methyltransferase 5 (PRMT5) has been identified as a potential therapeutic target for various cancer types. However, its role in regulating the hepatocellular carcinoma (HCC) transcriptome remains poorly understood. In this study, publicly available databases were employed to investigate PRMT5 expression, its correlation with overall survival, targeted pathways, and genes of interest in HCC. Additionally, we utilized in-house generated NGS data to explore PRMT5 expression in dysplastic nodules compared to hepatocellular carcinoma. Our findings revealed that PRMT5 is significantly overexpressed in HCC compared to normal liver, and elevated expression correlates with poor overall survival. To gain insights into the mechanism driving PRMT5 overexpression in HCC, we analyzed promoter CpG islands and methylation status in HCC compared to normal tissues. Pathway analysis of PRMT5 knockdown in the HCC cells revealed a connection between PRMT5 expression and genes related to the HIF1α pathway. Additionally, by filtering PRMT5-correlated genes within the HIF1α pathway and selecting up/downregulated genes in HCC patients, we identified Ras-related nuclear protein (RAN) as a target associated with overall survival. For the first time, we report that PRMT5 is implicated in the regulation of HIF1A and RAN genes, suggesting the potential prognostic utility of PRMT5 in HCC.

Maintenance of growth is important for sustaining yield under stress conditions. Hence, identification of genes involved in cell division and growth under abiotic stress is utmost important. Ras-related nuclear protein (Ran) is a small GTPase required for nucleocytoplasmic transport, mitotic progression, and nuclear envelope assembly in plants. In the present study, two Ran GTPase genes TaRAN1 and TaRAN2 were identified though genome-wide analysis in wheat (T. aestivum). Comparative analysis of Ran GTPases from wheat, barley, rice, maize, sorghum, and Arabidopsis revealed similar gene structure within phylogenetic clades and highly conserved protein structure. Expression analysis from expVIP platform showed ubiquitous expression of TaRAN genes across tissues and developmental stages. Under biotic and abiotic stresses, TaRAN1 expression was largely unaltered, while TaRAN2 showed stress specific response. In qRT-PCR analysis, TaRAN1 showed significantly higher expression as compared to TaRAN2 in shoot and root at seedling, vegetative, and reproductive stages. During progressive drought stress, TaRAN1 and TaRAN2 expression increase during early stress and restored to control level expression at higher stress levels in shoot. The steady-state level of transcripts was maintained to that of control in roots under drought stress. Under cold stress, expression of both the TaRAN genes decreased significantly at 3 h and became similar to control at 6 h in shoots, while salt stress significantly reduced the expression of TaRAN genes in shoots. The analysis suggests differential regulation of TaRAN genes under developmental stages and abiotic stresses. Delineating the molecular functions of Ran GTPases will help unravel the mechanism of stress induced growth inhibition in wheat. [ABSTRACT FROM AUTHOR]

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the potential association of five miRNA machinery gene polymorphisms (DICER1 rs3742330A>G, DROSHA rs10719T>C, RAN rs14035C>T, XPO5 rs11077A>C and DGCR8 rs417309G>A) with recurrent implantation failure (RIF), a clinical condition in which good-quality embryos repeatedly fail to implant following two or more in vitro fertilization cycles, and its associated risk factors in Korean women. Therefore, the present study performed genotype analysis and assessed the frequency of these miRNA gene polymorphisms in patients diagnosed with RIF (n=119) and randomly selected controls (n=210) with at least one live birth and no history of pregnancy loss. The DROSHA rs10719T>C and RAN rs14035C>T polymorphisms were identified to be significantly associated with decreased prevalence of RIF. Additionally, the DROSHA rs10719 TC and the RAN rs14035 CT genotypes were present at significantly lower frequencies in the RIF group than in the control group (adjusted odds ratio=0.550; 95% CI, 0.339-0.893; P=0.016; and adjusted odds ratio=0.590; 95% CI, 0.363-0.958; P=0.033, respectively). Furthermore, the combined RAN rs14035 CT+TT genotype was observed to be associated with decreased RIF prevalence (adjusted odds ratio=0.616; 95% CI, 0.386-0.982; P=0.042). Genotype combination analysis for the various miRNA polymorphisms revealed that the DROSHA TC genotype exhibited a highly significant negative association with RIF prevalence when combined with the RAN CT genotype (adjusted odds ratio=0.314; 95% CI, 0.147-0.673; P=0.003; false discovery rate-adjusted P=0.023). The present study revealed an association between the DROSHA rs10719 and RAN rs14035 3'UTR polymorphisms and decreased risk of RIF in Korean women, which suggests that these gene polymorphisms could represent potential markers for predicting RIF risk. [ABSTRACT FROM AUTHOR]

Small Ras‐like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras‐like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over‐expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA‐, RagB‐ and RagC‐dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity. WDR35, which causes genetic diseases including Sensenbrenner syndrome and seems to be a hedgehog signaling protein and a protein with a possible ciliary function, might be possible upstream regulator of RagA. WDR35 associates with RagA, RagB and RagC and might negatively influence mTORC1 activity. [ABSTRACT FROM AUTHOR]

Abstract The cell responses to different topographies have been observed for decades while the related mechanisms are still blurring. In the present study, we aim to test whether the Rac1, a member of Rho GTPases family that plays a crucial role in actin cytoskeleton dynamics, is involved in the micro/nano-topography (MNT) sensing and cell differentiation in rat bone marrow mesenchymal stem cells (rBMMSCs). Firstly, we found that the cell morphology was distinct on different topographies and the actin arrangement was significantly different, suggesting that the actin cytoskeleton was influenced by MNT. In the next, we found that the Rac1 expression was significantly improved on MNT surface and the actin cytoskeleton was severely impaired after Rac1 silencing. Then the osteogenic differentiation was performed to figure out whether the Rac1 was involved in cell differentiation that regulated by MNT surface. It was notable that the MNT could promote osteogenic differentiation, which was significantly inhibited after Rac1 silencing. Further analysis indicated that the ERK1/2 and p38, members of mitogen-activated protein kinase (MAPK) pathway, were regulated by Rac1. In conclusion, the Rac1 is involved in MNT sensing and may play a crucial role in cell differentiation regulation through the MAPK pathway. Graphical abstract Unlabelled Image Highlights • The Rac1 is upregulated on micro/nano-topography (MNT) surface in rat bone marrow mesenchymal stem cells (rBMMSCs). • The morphology and osteogenic differentiation of rBMMSCs on MNT surface are regulated by Rac1. • The mitogen-activated protein kinase (MAPK) pathway is activated on MNT surface through Rac1. [ABSTRACT FROM AUTHOR]

Abnormal expression of long noncoding RNAs (lncRNAs) often contributes to the unrestricted growth and invasion of cancer cells. lncRNA X-inactive specific transcript (XIST) expression is upregulated in several cancers; however, its underlying mechanism in osteosarcoma (OS) has not been elucidated. In the present study, we found that XIST expression was significantly increased in OS tissues and cell lines by LncRNA Profiler and qRT-PCR. The effects of XIST and miR-320b on OS cell proliferation and invasion were studied by MTT and Transwell invasion assays. The competing relationship between XIST and miR-320b was confirmed by luciferase reporter assay. Our results showed that XIST knockdown strikingly inhibited cell proliferation and invasion. Furthermore, XIST could directly bind to miR-320b and repress miR-320b expression. Moreover, XIST overexpression significantly relieved the inhibition on OS cell proliferation and invasion mediated by miR-320b overexpression, which involved the derepression of Ras-related protein RAP2B. We propose that XIST is responsible for OS cell proliferation and invasion and that XIST exerts its function through the miR-320b/RAP2B axis. Our findings suggest that lncRNA XIST may be a candidate prognostic biomarker and a target for new therapies in OS patients. [ABSTRACT FROM AUTHOR]

Breast cancer is the second leading cause of cancer-associated mortality in women worldwide. Strong evidence has suggested that Ran, which is a small GTP binding protein involved in the transport of RNA and protein across the nucleus, may be a key cellular protein involved in the metastatic progression of cancer. The present study investigated Ran gene expression in breast cancer tissue samples obtained from 140 patients who had undergone surgical resection for breast cancer. Western blot analysis of Ran in breast cancer tissues and paired adjacent normal tissues showed that expression of Ran was significantly increased in breast cancer tissues. Immunohistochemistry analyses conducted on formalin-fixed paraffin-embedded breast cancer tissue sections revealed that Ran expression was associated with tumor histological grade, nerve invasion and metastasis, vascular metastasis and Ki-67 expression (a marker of cell proliferation). Kaplan-Meier survival analysis showed that increased Ran expression in patients with breast cancer was positively associated with a poor survival prognosis. Furthermore, in vitro experiments demonstrated that highly migratory MDA-MB-231 cancer cells treated with Ran-si-RNA (si-Ran), which knocked down expression of Ran, exhibited decreased motility in trans-well migration and wound healing assays. Cell cycle analysis of Ran knocked down MDA-MB-231 cells implicated Ran in cell cycle arrest and the inhibition of proliferation. Furthermore, a starvation and re-feeding (CCK-8) assay was performed, which indicated that Ran regulated breast cancer cell proliferation. Taken together, the results provide strong in vitro evidence of the involvement of Ran in the progression of breast cancer and suggest that it could have high potential as a therapeutic target and/or marker of disease. [ABSTRACT FROM AUTHOR]

The Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein involved in a variety of intracellular signaling pathways that control diverse cellular functions. RanBPM interacts with proteins that are linked to various diseases, including Alzheimer’s disease (AD), schizophrenia (SCZ), and cancer. In this article, we define the characteristics of the scaffolding protein RanBPM and focus on its interaction partners in diverse physiological disorders, such as neurological diseases, fertility disorders, and cancer. [ABSTRACT FROM AUTHOR]

A ras-related nuclear protein (Ran) protein was obtained from Macrobrachium rosenbergii , named MrRan. Phylogenetic analysis results showed that MrRan was clustered in one group together with other crustaceans. Tissue distribution analysis revealed that MrRan was expressed mainly in gill, intestine and stomach, and expressed weakly in muscle. The MrRan expression levels in gill and hemocyte of prawns were significantly up-regulated after challenged by Spiroplasma eriocheiris . The copy number of S. eriocheiris in MrRan dsRNA injection group was significantly less than control groups during infection. Meanwhile, silencing MrRan obviously increased the survival rate of prawns. The subcellular localization experiment suggested that recombinant MrRan was mainly located in the nucleus, and relatively weak in the cytoplasm. Finally, over-expression in Drosophila S2 cell indicated that MrRan could increase copies of S. eriocheiris and decrease of cell viability. The present study suggested that MrRan participated in regulating the phagocytosis of S. eriocheiris in M. rosenbergii . [ABSTRACT FROM AUTHOR]

GEFs play a critical role in regulating Rac1 signaling. They serve as signaling nodes converting upstream signals into downstream Rac1-driven cellular responses. Through associating with membrane-bound Rac1, GEFs facilitate the exchange of GDP for GTP, thereby activating Rac1. As a result, Rac1 undergoes conformational changes that mediate its interaction with downstream effectors, linking Rac1 to a multitude of physiological and pathological processes. Interestingly, there are at least 20 GEFs involved in Rac1 activation, suggesting a more complex role of GEFs in regulating Rac1 signaling apart from promoting the exchange of GDP for GTP. Indeed, accumulating evidence implicates GEFs in directing the specificity of Rac1-driven signaling cascades, although the underlying mechanisms were poorly defined. Recently, through conducting a comparative study, we highlighted the role of 2 Rac-specific GEFs, Tiam1 and P-Rex1, in dictating the biological outcome downstream of Rac1. Importantly, further proteomic analysis uncovered a GEF activity-independent function for both GEFs in modulating the Rac1 interactome, which results in the stimulation of GEF-specific signaling cascades. Here, we provide an overview of our recent findings and discuss the role of GEFs as master regulators of Rac1 signaling with a particular focus on GEF-mediated modulation of cell migration following Rac1 activation. [ABSTRACT FROM AUTHOR]

Altered Rho GTPase signaling has been linked to many types of cancer. As many small G proteins, Rho GTPases cycle between an active and inactive state thanks to specific regulators that catalyze exchange of GDP into GTP (Rho-GEF) or hydrolysis of GTP into GDP (Rho-GAP). Recent studies have shown that alteration takes place either at the level of Rho proteins themselves (expression levels, point mutations) or at the level of their regulators, mostly RhoGEFs and RhoGAPs. Most reports describe Rho GTPases gain of function that may participate to the tumorigenesis processes. In contrast, we have recently reported that decreased activities of Cdc42 and Rac1 as well as decreased expression of 2 Rho-GEFs, FARP1 and ARHGEF1, correlate with pheochromocytomas, a tumor developing in the medulla of the adrenal gland (Croisé et al., Endocrine Related Cancer, 2016). Here we highlight the major evidence and further study the correlation between Rho GTPases activities and expression levels of ARHGEF1 and FARP1. Finally we also discuss how the decrease of Cdc42 and Rac1 activities may help human pheochromocytomas to develop and comment the possible relationship between FARP1, ARHGEF1 and the 2 Rho GTPases Cdc42 and Rac1 in tumorigenesis. [ABSTRACT FROM AUTHOR]

Influenza A virus is an important human pathogen causative of yearly epidemics and occasional pandemics. The ability to replicate within the host cell is a determinant of virulence, amplifying viral numbers for host-to-host transmission. This process requires multiple rounds of entering permissive cells, replication, and virion assembly at the plasma membrane, the site of viral budding and release. The assembly of influenza A virus involves packaging of several viral (and host) proteins and of a segmented genome, composed of 8 distinct RNAs in the form of viral ribonucleoproteins (vRNPs). The selective assembly of the 8-segment core remains one of the most interesting unresolved problems in virology. The recycling endosome regulatory GTPase Rab11 was shown to contribute to the process, by transporting vRNPs to the periphery, giving rise to enlarged cytosolic puncta rich in Rab11 and the 8 vRNPs. We recently reported that vRNP hotspots were formed of clustered vesicles harbouring protruding electron-dense structures that resembled vRNPs. Mechanistically, vRNP hotspots were formed as vRNPs outcompeted the cognate effectors of Rab11, the Rab11-Family-Interacting-Proteins (FIPs) for binding, and as a consequence impair recycling sorting at an unknown step. Here, we speculate on the impact that such impairment might have in host immunity, membrane architecture and viral assembly. [ABSTRACT FROM AUTHOR]

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states. [ABSTRACT FROM AUTHOR]

The nuclear lamina is essential for the proper structure and organization of the nucleus. Deregulation of A-type lamins can compromise genomic stability, alter chromatin organization and cause premature vascular aging. Here, we show that accumulation of the lamin A precursor, prelamin A, inhibits 53 BP1 recruitment to sites of DNA damage and increases basal levels of DNA damage in aged vascular smooth muscle cells. We identify that this genome instability arises through defective nuclear import of 53 BP1 as a consequence of abnormal topological arrangement of nucleoporin NUP153. We show for the first time that this nucleoporin is important for the nuclear localization of Ran and that the deregulated Ran gradient is likely to be compromising the nuclear import of 53 BP1. Importantly, many of the defects associated with prelamin A expression were significantly reduced upon treatment with Remodelin, a small molecule recently reported to reverse deficiencies associated with abnormal nuclear lamina. [ABSTRACT FROM AUTHOR]

RanBPM and RanBP10 are non-canonical members of the Ran binding protein family that lack the Ran binding domain and do not associate with Ran GTPase in vivo . Rather, they have been shown to be scaffolding proteins that are important for a variety of cellular processes, and both of these proteins contain a SPRY domain, which has been implicated in mediating protein–protein interactions with a variety of targets including the DEAD-box containing ATP-dependent RNA helicase (DDX-4). In this study, we have determined the crystal structures of the SPIa and the ryanodine receptor domain and of approximately 70 upstream residues (immediate upstream to SPRY motif) of both RanBPM and RanBP10. They are almost identical, composed of a β-sandwich fold with a set of two helices on each side located at the edge of the sheets. A unique shallow binding surface is formed by highly conserved loops on the surface of the β-sheet with two aspartates on one end, a positive patch on the opposite end, and a tryptophan lining at the bottom of the surface. The 20-mer peptide (residues 228–247) of human DDX-4, an ATP-dependent RNA helicase known to regulate germ cell development, binds to this surface with a K D of ~ 13 μM. The crystal structure of the peptide complex and the mutagenesis studies elucidate how RanBPM can recognize its interaction partners to function in gametogenesis. [ABSTRACT FROM AUTHOR]

Nucleotide-repeat expansions underlie a heterogeneous group of neurodegenerative and neuromuscular disorders for which there are currently no effective therapies. Recently, it was discovered that such repetitive RNA motifs can support translation initiation in the absence of an AUG start codon across a wide variety of sequence contexts, and that the products of these atypical translation initiation events contribute to neuronal toxicity. This review examines what we currently know and do not know about r epeat a ssociated n on-AUG (RAN) translation in the context of established canonical and non-canonical mechanisms of translation initiation. We highlight recent findings related to RAN translation in three repeat expansion disorders: CGG repeats in fragile X-associated tremor ataxia syndrome (FXTAS), GGGGCC repeats in C9orf72 associated amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and CAG repeats in Huntington disease. These studies suggest that mechanistic differences may exist for RAN translation dependent on repeat type, repeat reading frame, and the surrounding sequence context, but that for at least some repeats, RAN translation retains a dependence on some of the canonical translational initiation machinery. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease . [ABSTRACT FROM AUTHOR]

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation, and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase and was selected on the basis of yeast two-hybrid screen, GST pull-down assay and FRET measurements by fluorescence lifetime imaging microscopy. DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison with similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis. [ABSTRACT FROM AUTHOR]

Vitiligo is a depigmentation disorder characterized by loss of functional melanocytes of the skin epidermis. The pathogenesis of vitiligo remains elusive. The purpose of this study is to investigate the effects of basic fibroblast growth factor (bFGF) on melanocyte migration, including its biochemical mechanism using transwell assay in vitro. We found that melanocyte treated with bFGF showed a significant increase in migration and cytoskeletal rearrangement. These changes were associated with increased activation of PI3K/Akt, Rac1, FAK, JNK, and ERK. Likewise, reduction of PI3K/Akt, Rac1, FAK, JNK, and ERK activity using selective inhibitors or siRNA was associated with impediment of bFGF-induced melanocyte migration. In addition, activity of Rac1, FAK, and JNK was reduced in cells in which PI3K/Akt was inhibited, activity of FAK and JNK was reduced in cells in which the Rac1 was inhibited, and activity of JNK was reduced in cells in which the FAK was inhibited. Collectively, these data demonstrate that bFGF facilitated melanocyte migration via PI3K/Akt-Rac1-FAK-JNK and ERK signaling pathways. © 2016 IUBMB Life, 68(9):735-747, 2016 [ABSTRACT FROM AUTHOR]

Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes. [ABSTRACT FROM AUTHOR]

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-gua-nine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor la (HIF-lα) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-lα, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions - 1376 to - 1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1 /Rac1 pathway through HIF-lα-mediated transcriptional induction of CXCR4. [ABSTRACT FROM AUTHOR]

Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions. [ABSTRACT FROM AUTHOR]

In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild-type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, “fast cycling” mutant of Rac1, which is present in 5–10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a timedependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3= untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. [ABSTRACT FROM AUTHOR]

Rac1, a GTPase of the Rho subfamily, has a crucial role in cytoskeletal architecture, as well as the regulation of cell migration and growth. However, renal injury in mice with podocyte-specific deletion of Rac1 has yet to be elucidated fully due to conflicting findings. Herein, we identified a possible role for Rac1 in podocytes of streptozotocin- (STZ) induced diabetic mice. The urinary albumin/creatinine ratio (ACR) in the knockout (KO) group was significantly higher than that in the wild type (WT) group at any week of age. A more marked ACR increase was observed in STZ/KO group than STZ/WT group, although ACR did increase with weeks of age in both diabetic groups. The kidney sections from diabetic mice revealed a glomerular hypertrophy with mesangial expansion, but there was no appreciable difference in glomerular findings under a light microscope between STZ/WT and STZ/KO mice. However, an electron microscopy analysis revealed that regardless of the presence or absence of diabetes, both KO (KO and STZ/KO) groups had a higher rate of foot process effacement compared with both WT (WT and STZ/WT) groups. The expression levels of the slit diaphragm protein, podocin, was reduced with the induction of diabetes, and the levels in the STZ/KO group experienced a further reduction compared with the STZ/WT group. The number of WT1-positive cells in the STZ/KO group was more significantly decreased than that in the other three groups. In contrast, the numbers of cleaved caspase 3- and TUNEL-positive cells in the glomeruli of the STZ/KO group were more increased than those in the STZ/WT group. Thus, this study provides evidence that podocyte-specific deletion of Rac1 results in morphological alteration in podocytes, and that the induction of apoptosis or decreased expression of the slit diaphragm proteins by hyperglycemic stimuli are associated with the progression of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Summary A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting. [ABSTRACT FROM AUTHOR]

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway. [ABSTRACT FROM AUTHOR]

Ras-related nuclear protein (Ran) GTPase plays an important role in nucleo-cytoplasmic transportation of proteins and RNA, mitotic spindle assembly, microtubule assembly and nuclear envelope (NE) assembly. We previously identified the full-length cDNAs and a DNA of DlRan3A from longan ( Dimocarpus longan Lour.) somatic embryos and demonstrated its possible roles in cell activities during longan somatic embryogenesis (SE). However, thus far little is known of how Ran functions in the signaling pathways in plant embryos in response to changing environmental stimuli. To discover more biological roles of DlRan3A , we observed DlRan3A located in the nucleus and detected abundant accumulation of DlRan3A s in globular and cotyledon embryos during longan SE, which suggested its involvement in auxin signal pathways in longan early SE. The transcript level of the DlRan3A gene were also determined in longan embryogenic callus (EC) in response to different levels of exogenous phytohormones (indoleacetic acid (IAA), gibberellin A3 (GA 3 ), salicylic acid (SA), methyl jasmonate (MeJA), and ethephon (Eth)), salt, sucrose, prolonged culturing period and light quality treatments. IAA (1 mg/L), GA 3 (12 mg/L), SA (75 μm), MeJA (50 and 100 μm), and Eth (50 mg/L) modulate expression of DlRan3A to 1.3–1.4 folds, and the expression of DlRan3A was significantly affected by light quality, significantly induced to 2-fold by salt (10 g/L), 2.7-fold by sucrose (90 g/L) and was completely suppressed by prolonged cultivation (> 40 days). Deletion analysis suggested that both activation and repression regulatory elements co-exist in the DlRan3A promoter sequence and that the key cis-acting elements included ones in response to auxin, SA, MeJA, and stress. Promoter activities were induced to the highest level by IAA followed by SA, GA 3 and MeJA, while suppressed by ABA and Eth. Together, these results showed DlRan3A close involvement in phytohormones, light, and abiotic stress responsiveness during longan SE. [ABSTRACT FROM AUTHOR]

Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self-renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor-α (TNF-α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor-κB (NF-κB) signaling pathway. While previous studies showed that cells treated with TNF-α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF-α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF-α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2). Further, we revealed that TNF-α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β-catenin signaling pathway and liberate β-catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments. [ABSTRACT FROM AUTHOR]

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1-induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator . Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation. Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance. [ABSTRACT FROM AUTHOR]

Ca2+ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated CaV1.3 Ca2+ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca2+ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca2+ channels, resulting in reduced synaptic Ca2+ influx. Using superresolution microscopy, we found that Ca2+ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca2+ current, whereas the apparent Ca2+ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca2+ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca2+ channels at IHC AZs and are required for normal hearing. [ABSTRACT FROM AUTHOR]

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery. [ABSTRACT FROM AUTHOR]

The induction of suppressor of cytokine signalling (SOCS)3 expression context dependently involves regulation of SOCS3 transcript stability as previously demonstrated for MAPK activated protein kinase (MK)2-dependent regulation of SOCS3 expression by TNFα (Ehlting et al., 2007). In how far the IL-6-type cytokine OSM, which in contrast to IL-6 is a strong activator of p38 MAPK /MK2 signalling, also involves regulation of transcript stability and activation of MK2 to induce SOCS3 expression is unclear. In contrast to IL-6, OSM induces SOCS3 expression in murine fibroblasts and in primary human and murine hepatocytes, but not in macrophages because the latter lack the OSM receptor (OSMR)β subunit. Evidence is provided that regulation of OSM-induced expression of SOCS3 involves MEK1- and Erk1/2-mediated stabilization of the SOCS3 transcript. Consistently, OSM-induced stabilization of the SOCS3 transcript is impaired in the presence of inhibitors that specifically block activation of MEK1/2 (U0126) and ERK1/2 (FR180204) or upon knock-down of ERK1/2 expression using specific siRNA. As a potential target site that integrates the stability regulating effect of OSM and OSM-induced activation of MEK1/2 and ERK1/2 a region containing three copies of a pentameric AUUUA motif located within position 2422 and 2541 in closed proximity to the 3′ UTR of the SOCS3 transcript has been identified. Unexpectedly, activation of the p38 MAPK /MK2 pathway, which apart from STAT3 and ERK1/2, is also strongly activated by OSM in human and murine hepatocytes and murine fibroblasts is dispensable for stabilization of the SOCS3 transcript as suggested from inhibitor studies using the p38 MAPK inhibitor SB203580 or from the analysis of MK2-deficient hepatocytes. However, analysis of MK2-deficient macrophages and hepatocytes revealed that, although MK2 is dispensable for regulation of OSM-induced SOCS3 expression, MK2 is essential for LPS-induced OSM production in macrophages and limits the overall availability of the OSMRβ subunit in hepatocytes. Thus MK2 plays a role for the induction and sensing of OSM-mediated intercellular signalling between macrophages and hepatocytes during LPS-induced inflammation. [ABSTRACT FROM AUTHOR]

The article discusses a report on the function of a Kinesin-like protein KIF1C which transports the Ras-related protein Rab6A & can influence the Golgi complex organization. It also focuses on changes after the KIF1C depletion such as, slow protein delivery to cell surface, interfering with the vesicle motility & triggers Golgi fragmentation. In addition KIF1C can protect the Golgi membranes from fragmenting the cells which lack an intact micro-tubule network.

The article discusses a study related to the type of karyopherin protein that regulates the permeability of the nuclear pore complex during nuclear transport, and discusses the functions of importin beta and RanGTP. Topics include transport of inert cargos and formation of multivalent complexes. The study concludes that the interaction between importin beta and NUP153 proteins at the face of nuclear core complex forms a Ran-regulated mesh, which in turn modulates the permeability.

Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration. [ABSTRACT FROM AUTHOR]

We performed a comparative study between two human metastatic melanoma cell lines (A375 and 526), and melanocytes (FOM78) by gene expression profiling and pathway analysis, using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software. Genes involved in Ran signaling were significantly over-represented (p ≤ 0.001) and up-regulated in melanoma cells. A melanoma-associated molecular pathway was identified, where Ran, Aurora Kinase A (AurkA) and TERT were up-regulated, while c-myc and PTEN were down-regulated. A consistent high Ran and AurkA gene expression was detected in about 48% and 53%, respectively, of 113 tissue samples from metastatic melanoma patients. AurkA down-regulation was observed in melanoma cells, by Ran knockdown, suggesting AurkA protein is a Ran downstream target. Furthermore, AurkA inhibition, by exposure of melanoma cells to MLN8054, a specific AurKA inhibitor, induced apoptosis in both melanoma cell lines and molecular alterations in the IPA-identified molecular pathway. These alterations differed between cell lines, with an up-regulation of c-myc protein level observed in 526 cells and a slight reduction seen in A375 cells. Moreover, Ran silencing did not affect the A375 invasive capability, while it was enhanced in 526 cells, suggesting that Ran knockdown, by AurkA down-regulation, resulted in a Ran-independent enhanced melanoma cell invasion. Finally, AurK A inhibition induced a PTEN up-regulation and its action was independent of B-RAF mutational status. These findings provide insights relevant for the development of novel therapeutic strategies as well as for a better understanding of mechanisms underlying therapy resistance in melanoma. [ABSTRACT FROM AUTHOR]

Novel series of Farnesylthiosalicylic acid-diamine/phenylpropenoic acid hybrids were designed and synthesized. Their in vitro growth inhibitory assays showed that most compounds displayed strong antiproliferation activity against seven cancer cells. Especially, the new hybrid 12f, by the conjugation of 10a with ferulic acid, could selectively suppress the proliferation of tumor cells and display significantly lower toxicities to normal cells than its intermediate 10a. Furthermore, 12f dose-dependently induced SMMC-7721 cell apoptosis. Additionally, our observations demonstrated that 12f inhibited both Ras-related signaling and phosphorylated NF- κB synergistically, which may be advantageous to the strong antitumor activities of 12f. Our findings suggest that these novel hybrids may hold a great promise as therapeutic agents for the intervention of human cancers. [ABSTRACT FROM AUTHOR]

Summary Background The γ-tubulin ring complex (γTuRC) is a multisubunit complex responsible for microtubule (MT) nucleation in eukaryotic cells. During mitosis, its spatial and temporal regulation promotes MT nucleation through different pathways. One of them is triggered around the chromosomes by RanGTP. Chromosomal MTs are essential for functional spindle assembly, but the mechanism by which RanGTP activates MT nucleation has not yet been resolved. Results We used a combination of Xenopus egg extracts and in vitro experiments to dissect the mechanism by which RanGTP triggers MT nucleation. In egg extracts, NEDD1-coated beads promote MT nucleation only in the presence of RanGTP. We show that RanGTP promotes a direct interaction between one of its targets, TPX2, and XRHAMM that defines a specific γTuRC subcomplex. Through depletion/add-back experiments using mutant forms of TPX2 and NEDD1, we show that the activation of MT nucleation by RanGTP requires both NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex through a TPX2-dependent mechanism. Conclusions The XRHAMM-γTuRC complex is the target for activation by RanGTP that promotes an interaction between TPX2 and XRHAMM. The resulting TPX2-RHAMM-γTuRC supracomplex fulfills the two essential requirements for the activation of MT nucleation by RanGTP: NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex onto a TPX2-dependent scaffold. Our data identify TPX2 as the only direct RanGTP target and NEDD1 as the only Aurora A substrate essential for the activation of the RanGTP-dependent MT nucleation pathway. [ABSTRACT FROM AUTHOR]

Cdc42 is a Ras-related small GTP-binding protein. A previous study has shown that Cdc42 binding to the ? subunit of the coatomer protein complex (?COP) is essential for Cdc42-regulated cellular transformation, but the molecular mechanism involved is not well understood. Here, we demonstrate that constitutively-active Cdc42 binding to ?COP induced the accumulation of epithelial growth factor receptor (EGFR) in the cells, sustained EGF-stimulated extracellular signal-regulated kinase (ERK), JUN amino-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) signaling and promoted cell division. Moreover, constitutive Cdc42 activity facilitated the nuclear translocation of EGFR, and this indicates a novel mechanism through which Cdc42 might promote cellular transformation. [ABSTRACT FROM AUTHOR]

Fucoid zygotes use environmental vectors, including sunlight, to initiate a growth axis a few hours after fertilization. The first division is then transversely oriented by the growth axis, producing daughter cells of distinct fates. The tip growing rhizoid cell gives rise to the holdfast, anchoring the alga to the intertidal substratum, while the opposite thallus cell mainly generates the photosynthetic and reproductive stipe and fronds. Elaboration of this simple growth axis thus establishes the basic body plan of the adult; and elucidating the mechanisms responsible for formation of the growth axis is paramount to understanding fucoid morphogenesis. Recent studies have culminated in a model whereby sunlight, and perhaps other environmental cues, activate the signaling protein Rac1 at the rhizoid pole. Here it sets in motion nucleation of a patch of actin filaments that in turn, targets ions, proteins, and cellular processes to the future growth site. At germination, Rac1 initiates morphogenesis by inducing transformation of the patch of actin filaments to a structure that delivers vesicles to the growing tip, and a few hours later orients the spindle and cytokinetic plate. [ABSTRACT FROM AUTHOR]

For more than 15 years, TPX2 has been studied as a factor critical for mitosis and spindle assembly. These functions of TPX2 are attributed to its Ran-regulated microtubule-associated protein properties and to its control of the Aurora A kinase. Overexpressed in cancers, TPX2 is being established as marker for the diagnosis and prognosis of malignancies. During interphase, TPX2 resides preferentially in the nucleus where its function had remained elusive until recently. The latest finding that TPX2 plays a role in amplification of the DNA damage response, combined with the characterization of TPX2 knockout mice, open new perspectives to understand the biology of this protein. This review provides an historic overview of the discovery of TPX2 and summarizes its cytoskeletal and signaling roles with relevance to cancer therapies. Finally, the review aims to reconcile discrepancies between the experimental and pathological effects of TPX2 overexpression and advances new roles for compartmentalized TPX2. [ABSTRACT FROM AUTHOR]

Aims Rac1, Pak1 and Rock1 are indicators related to gastric cancer invasion and metastasis, but few reports discuss all three kinds of protein in research on gastric cancer invasion and metastasis. The aim of this study was to investigate the expression and clinical significance of Rac1, Pak1 and Rock1 in gastric carcinoma. Methods Rac1, Pak1 and Rock1 expression in 158 cases of gastric carcinoma were investigated via immunohistochemical staining and clinical analysis. Results The positive expression rates of Rac1, Pak1 and Rock1 in normal tissue, intraepithelial neoplastic tissues and gastric carcinoma showed an increasing trend ( P < 0.05). Their expression in lymph node metastasis was significantly higher than in patients with lymph-node metastasis than in those without lymph nodes metastasis ( P < 0.05). Their expression in tumor ( TNM stages III and IV) were significantly higher than that in stages I and II ( P < 0.05). Rac1, Pak1 and Rock1 expression did not differ significantly with patients' sex ( P > 0.05). Conclusion Positive rates of Rac1, Pak1 and Rock1 expression in normal tissue, dysplasia and gastric carcinoma show an increasing trend and are correlated with tumor lymph node metastasis and TNM stage. Rac1, Pak1 and Rock1 may be important biomarkers of gastric carcinoma invasion and metastasis. [ABSTRACT FROM AUTHOR]

Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-a2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network. [ABSTRACT FROM AUTHOR]

Highlights: [•] Ran took great effects on the regulation of phagocytosis against DCV infection. [•] Ran played an important role throughout the entire phagocytic process. [•] The study contributed a novel aspect of Ran protein in innate immunity of animal. [Copyright &y& Elsevier]

Highlights: [•] Overexpression of Ran in pancreatic cancer was correlated with histological grade. [•] Downregulation of Ran could induce cell apoptosis and inhibit cell proliferation. [•] The effects were mediated by cell cycle proteins, Survivin and cleaved Caspase-3. [Copyright &y& Elsevier]

Abstract: The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. [Copyright &y& Elsevier]