The Rho exchange factors Vav2 and Vav3 favor skin tumor initiation and promotion by engaging extracellular signaling loops.

The catalytic activity of GDP/GTP exchange factors (GEFs) is considered critical to maintain the typically high activity of Rho GTPases found in cancer cells. However, the large number of them has made it difficult to pinpoint those playing proactive, nonredundant roles in tumors. In this work, we have investigated whether GEFs of the Vav subfamily exert such specific roles in skin cancer.

Fission yeast TORC1 prevents eIF2α phosphorylation in response to nitrogen and amino acids via Gcn2 kinase.

Serine 51 phosphorylation of the eukaryotic initiation factor-2α (eIF2α) is an important mechanism involved in blocking general protein synthesis in response to diverse types of stress. In fission yeast, three kinases (Hri1, Hri2 and Gcn2) can phosphorylate eIF2α at serine 51. In this study, we show that Tor2, as part of the TORC1 complex, prevents the phosphorylation of eIF2α in cells growing in the presence of nitrogen and amino acids.

The rho exchange factors vav2 and vav3 control a lung metastasis-specific transcriptional program in breast cancer cells.

The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, and many of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs.

Rac-ing to the plasma membrane: the long and complex work commute of Rac1 during cell signaling.

The functional cycle of the Rac1 GTPase involves a large number of steps, including post-translational processing, cytosolic sequestration by RhoGDIs, translocation to specific subcellular localizations, activation by GDP/GTP exchange, inactivation by GTP hydrolysis, and re-formation of cytosolic Rac1/RhoGDI inhibitory complexes. Here, we summarize the current knowledge about the regulation of those steps. In addition, we discuss a recently described, cytoskeletal-dependent feed-back loop that favors the efficient translocation and activation of Rac subfamily proteins during cell signaling.

Intratumoral stages of metastatic cells: a synthesis of ontogeny, Rho/Rac GTPases, epithelial-mesenchymal transitions, and more.

Metastasis is one of the clinical parameters that has a strong negative influence on the prognosis of cancer patients. In recent years, significant advances have furthered our understanding of this process at the molecular and biological levels. This paper will discuss recent discoveries relating to the earliest, intra-tumoral stages of metastasis in cancer cells, specifically focusing on: (i) the development of metastatic traits during primary tumorigenesis; (ii) intrinsic and extrinsic cancer cell programs associated with malignant traits; (iii) the intra-tumoral migration patterns of cancer cells and the dynamic roles played by the Rho/Rac GTPases and epithelial-mesenchymal transitions in this process; and (iv) the genetic strategies used by metastatic cancer cells to promote intra-tumoral cell migration and their subsequent escape to peripheral tissues.

The Vam6 and Gtr1-Gtr2 pathway activates TORC1 in response to amino acids in fission yeast.

The Rag family of GTPases has been implicated in the TORC1 activation in Drosophila and in mammalian cells in response to amino acids. We have investigated the role of the Rag GTPases Gtr1 and Gtr2 in TORC1 regulation in Schizosaccharomyces pombe. Fission yeast Gtr1 and Gtr2 are non-essential proteins that enhance cell growth in the presence of amino acids in the medium.

New models towards assessing anti-cancer therapeutics.

Cancer is the subject of intense research around the world, but many questions about how the disease works remain unanswered. How exactly does cancer start and how do tumours grow? In fact, at present there are ten times more anticancer drugs being tested in clinical trials than there were 15 years ago. However, many of the new anticancer agents are predicted to show clinical benefit in only small subpopulations of patients.

New polyether triterpenoids from Laurencia viridis and their biological evaluation.

The red seaweed Laurencia viridis is a rich source of secondary metabolites derived from squalene. New polyethers, such as iubol (2), 22-hydroxy-15(28)- dehydrovenustatriol (3), 1,2-dehydropseudodehydrothyrsiferol (4), and secodehydrothyrsiferol (5) have been isolated and characterized from this alga. The structures were determined through the interpretation of NMR spectroscopic data and the relative configuration was proposed on the basis of NOESY spectrum and biogenetic considerations.

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes.

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet.

The Ddc2/ATRIP checkpoint protein monitors meiotic recombination intermediates.

During meiosis, accurate segregation of intact chromosomes is essential for generating healthy gametes. Defects in recombination and/or chromosome synapsis activate the pachytene checkpoint, which delays meiotic cell cycle progression to avoid aberrant chromosome segregation and formation of defective gametes. Here, we characterize the role of the conserved DNA damage checkpoint protein Ddc2/ATRIP in this meiotic surveillance mechanism.

A transcriptional cross-talk between RhoA and c-Myc inhibits the RhoA/Rock-dependent cytoskeleton.

The GTPase RhoA and the transcriptional factor c-Myc are closely intertwined in cancer cells. Although this cross-talk results in potent synergistic effects that favor the transformed phenotype of cancer cells, recent results from our laboratory indicate that c-Myc also participates in a negative feed-back loop that blocks specific RhoA signaling branches connected to the induction of stress fibers, focal adhesions and actomyosin contractility. Using microarray analysis, we have unveiled a RhoA/c-Myc-dependent gene signature in charge of this negative cross-talk.

Ribosome synthesis-unrelated functions of the preribosomal factor Rrp12 in cell cycle progression and the DNA damage response.

Given the high metabolic cost required to generate ribosomes, it has been assumed that proteins involved in ribosome synthesis might establish functional cross talk with other intracellular processes to efficiently couple ribosome production and cell growth. However, such interconnections have remained elusive due to the difficulty in separating the intra- and extraribosomal roles of ribosome biogenesis factors. Using a yeast functional screen, I have discovered that Rrp12, a conserved protein involved in ribosome maturation and export, plays roles in the cell cycle and the DNA damage response.

Strategies for isolating constitutively active and dominant-negative pheromone receptor mutants in yeast.

Mating pheromone receptors of the yeast Saccharomyces cerevisiae are useful models for the study of G protein-coupled receptors. The mating pheromone receptors, Ste2 and Ste3, are not essential for viability so they can be readily targeted for analysis by a variety of genetic approaches. This chapter will describe methods for identification of two kinds of mutants that have been very informative about the mechanisms of receptor signaling: constitutively active mutants and dominant-negative mutants.

Upregulation of the PRB1 gene in the Saccharomyces cerevisiae rim101Delta mutant produces proteolytic artefacts that differentially affect some proteins.

Proteolytic degradation during protein processing in yeast is usually prevented by the addition of protease inhibitors or strict cooling of the samples. In this report we show that, while these precautions are sufficient for some strains, they are clearly insufficient for others. Specifically, we show that the stability of some proteins, such as Slt2p or Chs4p, but not others, is severely compromised in the rim101Delta mutant due to the upregulation of the PRB1 gene, which leads to higher levels of proteinase B activity.

Lipid rafts and clusters of apoptotic signaling molecule-enriched rafts in cancer therapy.

There is an imperative need to overcome the rather poor outcomes of current cancer chemotherapy with further development of novel targets and drugs. Direct activation of the cancer cell apoptotic machinery constitutes an appealing approach. Recent findings have shown that apoptosis can be triggered by co-aggregation of lipid rafts with death receptors and downstream signaling molecules, thus facilitating their interactions to convey apoptotic signals.

A transcriptional cross-talk between RhoA and c-Myc inhibits the RhoA/Rock-dependent cytoskeleton.

The GTPase RhoA participates in a number of cellular processes, including cytoskeletal organization, mitogenesis and tumorigenesis. We have previously shown that the transforming activity of an oncogenic version of RhoA (Q63L mutant) was highly dependent on the transcriptional factor c-Myc. In contrast to these positive effects in the RhoA route, we show here that c-Myc affects negatively the F-actin cytoskeleton induced by RhoA(Q63L) and its downstream effector, the serine/threonine kinase Rock.

The Rho/Rac exchange factor Vav2 controls nitric oxide-dependent responses in mouse vascular smooth muscle cells.

The regulation of arterial contractility is essential for blood pressure control. The GTPase RhoA promotes vasoconstriction by modulating the cytoskeleton of vascular smooth muscle cells. Whether other Rho/Rac pathways contribute to blood pressure regulation remains unknown.

Identification of the Rock-dependent transcriptome in rodent fibroblasts.

Rock proteins are Rho GTPase-dependent serine/ threonine kinases with crucial roles in F-actin dynamics and cell transformation. By analogy with other protein kinase families, it can be assumed that Rock proteins act, at least in part, through the regulation of gene expression events. However, with the exception of some singular transcriptional targets recently identified, the actual impact of these kinases on the overall cell transcriptome remains unknown.

Role of chimaerins, a group of Rac-specific GTPase activating proteins, in T-cell receptor signaling.

Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that alpha2- and beta2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling.

RasGRF2, a guanosine nucleotide exchange factor for Ras GTPases, participates in T-cell signaling responses.

The Ras pathway is critical for the development and function of T lymphocytes. The stimulation of this GTPase in T cells occurs primarily through the Vav1- and phospholipase C-gamma1-dependent activation of RasGRP1, a diacylglycerol-responsive Ras GDP/GTP exchange factor. Here, we show that a second exchange factor, RasGRF2, also participates in T-cell signaling.

The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.

The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome.

GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo.

Rho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family.

Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases.

We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially.

Involvement of the Rho/Rac family member RhoG in caveolar endocytosis.

We show here that the GTPase RhoG is involved in caveolar trafficking. Wild-type RhoG moves sequentially to the plasma membrane, intracellular vesicles, and the Golgi apparatus along markers of this endocytic pathway. Such translocation is associated with changes in RhoG GDP/GTP levels and is highly dependent on lipid raft integrity and on the function of the GTPase dynamin2.