C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

An Eph receptor regulates integrin activity through R-Ras

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.

The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and Is Important for the Proper Aggregation of Developing Cells

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.

Papel de C3G y p38α en la regulación de la migración, invasión tumoral en carcinoma de colon. Función de fibulina 3

Colorectal cancer (CRC) represents the third most common cancer type and the second leading cause of cancer-related death in the western world. CRC results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into adenocarcinoma (Lao and Grady 2011), affecting several genes such as Apc, K-ras, dcc/Smad4 and p53 or DNA mismatch repair genes (Pancione et al. 2012). p38 MAPKs are a subfamily of Serine-Threonine kinases activated by different stimuli that control fundamental cellular processes such as cell growth, proliferation, differentiation, migration and apoptosis (Dhillon et al. 2007, Nebreda and Porras 2000, Wagner and Nebreda 2009). There are four p38 MAPKs isoforms in mammals: α, β, δ and γ. p38α MAPK is ubiquitously expressed and is the most abundant isoform (Cuenda and Rousseau 2007). p38α is involved in the regulation of many cellular functions, among them, cell migration and invasion. In cancer, it can act as either a promoter or a suppressor of tumor growth, playing different roles during tumor progression (del Barco Barrantes and Nebreda 2012). C3G is a guanine nucleotide exchange factor (GEF) mainly for the Ras family members: Rap1 (Gotoh et al. 1995) and R-Ras (Gotoh et al. 1997), but it can also act through GEF independent mechanisms. C3G regulates several cellular functions such as cell death, adhesion, migration and invasion (Radha et al. 2011). In collaboration with Dr. Carmen Guerrero’s group (Centro del Investigación del Cáncer de Salamanca), our group has found a new functional relationship between C3G and p38α MAPK involved in the regulation of cell death in MEFs (Gutierrez-Uzquiza et al. 2010) and in the chronic myeloid leukemia (CML) K562 cell line (Maia et al. 2009). Moreover, C3G and p38α act through a common regulatory pathway to control cell adhesion in K562 cells regulating focal adhesion proteins (Maia et al. 2013)...

al- Risālah al-ūlá min rasāʼil al-Tājūrī fī ʻilm al-mīqāt wa-al-falak : manuscript, 1635

Written in one column, 25 lines per page, in black and red.

al- Risālah al-thānīyah min rasāʼil al-ʻAllāmah al-Shaykh ʻAbd al-Raḥmān al-Tājūrī : manuscript, [ca. 1635]

Written in one column, 25 lines per page, in black and red.

Rasāʼil mufīdah fī bayān mawḍūʻ ʻilm al-tafsīr wa-taʻrīfihi wa-istimdādihi wa-ghāyatihi / lil-Imām al-ʻĀlim al-ʻAllāmah mufīd al-ṭālibīn Muḥammad ibn Khalīfah ibn Ṣadr al-mudarrisīn al-Shaykh Saʻd al-Dīn al-Marḥūmī al-Shawbarī : manuscript, undated

Written in one column, 21 lines per page, in black and red.

Helaethrwydd o ras i'r pennaf o bechaduriaid: mewn ffyddlon hanes o fywyd a marwolaeth y Parch.

Mode of access: Internet.

H-ras activation is an early event in the ptaquiloside-induced carcinogenesis: Comparison of acute and chronic toxicity in rats

Bracken fern (Pteridium spp.) produces cancer of the urinary bladder and oesophagus in grazing animals and is a suspected human carcinogen, The carcinogenic principle ptaquiloside (PT), when activated to a dienone (APT), forms DNA adducts which eventually leads to tumor. Two groups of female Sprague-Dawley rats were given a chronic dose of 3 mg APT weekly for 10 weeks either by intravenous (iv) tail vein or by intragastric (ig) route, A third group was given a weekly dose of 6 mg of APT for 3 weeks by the ig route corresponding to acute dosing. Both chronic iv and ig dosed animals showed ischemic tubular necrosis in the kidney but only iv dosed animals developed adenocarcinomas of the mammary glands. Acutely dosed ig animals produced apoptotic bodies in the liver, necrosis of blood cell precursors in the bone marrow and ischemic tubular necrosis in the kidney but they did not develop tumors, No mutations were found in the H-ras and p53 genes in the mammary glands of either the ig rats or the tumor-bearing iv rats. However, the mammary glands of a fourth group of rats, which received APT by iv and killed before tumor development, carried Pu to Pu and Pu to Py double mutations in codons 58 and 59 of H-ras. This study indicates that the route of administration plays a role in the nature of the disease expression from ptaquiloside exposure. In addition to confirming the role of APT in the PT-induced carcinogenesis our finding suggests that activation of H-ras is an early event in the PT-carcinogenesis model. (C) 1998 Academic Press.

PCR bias toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Caveolin and Ras function

Experimental protocols that allow confident assignment of signaling proteins to specific subdomains of the plasma membrane are essential for a full understanding of the complexities of signal transduction. This is especially relevant for Ras proteins, where the different membrane anchors of the Ras isoforms target them to functionally distinct microdomains that in turn allow quantitatively different signal outputs from otherwise highly homologous proteins. The methods outlined in this chapter, in addition to being invaluable in addressing Ras function, should also have wide utility in the study of many mammalian signal transduction pathways.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.