The Ras superfamily of small GTPases comprises a group of molecular switches that regulate an astonishing diversity of cellular functions. A deep understanding of mitogenesis, cytoskeletal organization, vesicle traffic, and nuclear transport now requires the inclusion of the small GTPases as essential components of the molecular machines that drive these processes. The rich complexity of the control mechanisms involved is evidenced by the recent discoveries of GTPase cascades, multiple downstream effectors, and interconnected networks of GTPase-regulated protein kinase cascades. The 1995 FASEB Summer Conference at Snowmass Village, Colorado, on the Ras GTPase superfamily provided testimony to the broad impact that the study of these proteins continues to exert on cell biology.

When oxygen demand is greater than oxygen supply, cells need to rapidly adjust their metabolism in order for the tissue to survive. Oxygen sensing by an organism influences a host of processes including growth, development, metabolism, pH homeostasis, and angiogenesis. Hypoxia also contributes to a wide number of human diseases including vascular disease, inflammatory conditions and cancer. Recently, major advances have been made in understanding the response of cells and tissues to hypoxia with the goal of providing mechanistic insight and novel therapeutic targets. In this article we review both the normal biological effects of hypoxia as well as the alterations that occur in specific disease conditions with an emphasis on the cell signaling and gene transcription mechanisms that underlie the changes associated with chronic hypoxia. Comparisons of studies in the fields of cardiac ischemia and tumor angiogenesis reveal the complexities within the microenvironment that control responses to hypoxia. It is clear that more interaction between researchers in these fields will improve the development of therapies that either promote or prevent hypoxic responses.

The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore. RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran. RanBP1 also promotes the binding of Ran to karyopherin beta (also called importin beta and p97) and is a co-stimulator of RanGAP activity. Yeast karyopherin beta inhibits the GTP hydrolysis by Ran catalyzed by RanGAP. To further define the roles of RanBP1 and karyopherin beta in Ran function, we explored the effects of RanBP1 and karyopherin beta on mammalian proteins known to regulate Ran. Like RanBP1, karyopherin beta prevented the release of GTP from Ran stimulated by RCC1 or EDTA. As with the yeast protein, mammalian karyopherin beta completely blocked RanGAP activity. However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity. Kinetic analysis of the effects on RanGAP activity by karyopherin beta and RanBP1 revealed a combination of competitive and noncompetitive interactions. Solution binding assays confirmed the ability of RanBP1 to associate with Ran and karyopherin beta in a ternary complex, and RanBP1 binding was not competed out by the addition of karyopherin beta. These results demonstrate that RanBP1 and karyopherin beta interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to karyopherin beta.

Abstract. RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and 13-importin/13-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD. In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.T RAFFIC between the nucleus and the cytoplasm occurs through nuclear pore complexes in the nuclear membrane. Proteins containing a nuclear localization signal (NLS) 1 are actively transported into the nucleus by the nuclear import machinery. Several components involved in this process have been identified, and nuclear protein transport can be reconstituted in permeabilized cells that have been depleted of cytoplasm (for review see . Nuclear protein export is not nearly as well understood, but the recent identification of nuclear export signals (NESs) may provide insight into the molecular basis for this process (for review see Gerace, 1995). NESs have been identified in three unrelated proteins thus far" protein kinase inhibitor (PKI) (Wen et al., 1995), HIV-1 Rev (Fischer et al., 1995), and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Michael et al., 1995). The PKI and HIV-1 Rev NESs show limited similarity to one another, but the hnRNP A1 NES is distinctly different. All three proteins have been shown to shuttle between the nucleus and the cytoplasm and to mediate the export of other macromolecules.

Oxidants in cigarette smoke and generated from asbestos fibers activate mitogen-activated protein kinase (MAPK) signaling cascades in lung epithelial cells in vitro and in vivo. These signaling pathways lead to the enhanced ability of Jun and Fos family members (i.e., components of the activator protein [AP]-1 transcription factor) to activate transcription of a number of AP-1-dependent target genes involved in cell proliferation or death, differentiation, and inflammation. Research by the Basbaum laboratory has been critical in showing that mucin transcription in response to cigarette smoke and gram-positive bacteria is mediated through activation of the epidermal growth factor receptor and MAPK cascades. Work from our laboratories supports the concept that MAPK signaling and AP-1 transactivation by cigarette smoke and asbestos may synergize in lung epithelial cell injury, compensatory proliferation of lung epithelial cells, and carcinogenesis, supporting a mechanistic framework for the striking increases in lung cancer incidence in asbestos workers who smoke. Targeting of MAPKs and inter-related signaling cascades may be critical to the prevention of lung cancers and control of mucin overproduction in a number of lung diseases including asthma, cystic fibrosis, chronic bronchitis, and chronic obstructive pulmonary disease.Keywords: activator protein-1; asbestos; cigarette smoke; epidermal growth factor receptor; extracellular signal regulated kinases Reactive oxygen species (ROS) and reactive nitrogen species are mediators of cell signaling in airway and pulmonary epithelial cells by a variety of toxic agents, including asbestos and silica, cigarette smoke, airborne particulate matter, diesel exhaust, and ozone. These signaling cascades are under intense investigation by a number of laboratories because of their links to exacerbation of epithelial cell injury and proliferation, cell survival and chemoresistance, altered differentiation, and production of chemokines or cytokines mediating inflammation.The mitogen-activated protein kinase (MAPK) cascades consist of serine threonine kinases that are sequentially phosphorylated by upstream kinases (MAPKKK, MAPKK) and subdivided into three major pathways: extracellular signal-regulated kinases (ERKs), c-Jun-NH 2 -terminal kinases (JNKs 1, 2, and 3) (also referred to as stress-activated protein kinases), and p38 kinases (1-3) (Figure 1). MAPK cascades can be initiated by activation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) or other factors stimulating phosphorylation of upstream MAPKKK and MAPKK. Because the MAPK cascades involve sequential phosphorylation of protein kinases, factors inhibiting the phosphatases that normally check these pathways also cause net increases in the phosphorylation (i.e., activation) of MAPK proteins.Specific MAPKs control the activation of fos and jun family protooncogenes and their protein products (c-Jun, Jun B, Jun D, c-Fos, Fos B, Fra-1, and Fra-2) that are also known as AP-1 family members. The...

Ran/TC4 is a member of the Ras superfamily of GTPases. It is unusual in being predominantly nuclear and because it possesses an acidic -DEDDDL sequence instead of a consensus prenylation domain at the C terminus. Ran is required for nuclear protein import and cell cycle progression, and has been implicated in mRNA processing and export and DNA replication. The inhibition of cell cycle progression by a dominant gain-of-function mutant of Ran has been shown to be abrogated by removal of the -DEDDDL sequence, suggesting that this domain is essential for Ran function. We demonstrate here that the -DEDDDL sequence stabilizes GDP binding to Ran, and that the domain is required for high affinity interaction with a Ran-binding protein, HTF9A/RanBP1. HTF9A functions as a co-stimulator of Ran-GAP (GTPase activating protein) activity on wild-type Ran, but in the absence of the acidic C terminus of Ran, HTF9A behaves as a Ran-GAP inhibitor. An antibody directed against the C-terminal region preferentially recognizes the GTP-bound form of Ran, suggesting that this domain undergoes a nucleotide-dependent conformational change. The results suggest that the acidic C-terminal domain is important in modulating the interaction of Ran with regulatory factors, and implicate Ran-binding proteins in mediating the effects of Ran on cell cycle progression.

Abstract. The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (tl/2 ~'~ 5 min). Cotransfection with epitopetagged, wild-type Ran led to expression of HAl-Ran that was ~l.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (AC) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following microinjection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP-and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.

Elevation of nitric oxide (NO) release from the vascular endothelium may contribute to some of the gender-associated differences in coronary artery function. The mechanisms by which gender affects NO release from the endothelium of coronary arteries are not known. In this study, endothelial function was examined in pressurized coronary arteries from female and male rats. Diameter and endothelial cell intracellular Ca2+concentration ([Ca2+]i) in intact arteries, as well as enzymatic activity of endothelial constitutive nitric oxide synthase (ecNOS) in arterial lysates, was measured. Elevation of intravascular pressure to 60 mmHg constricted coronary arteries from female animals less than coronary arteries from male animals (18% and 31% constriction, respectively). The increased arterial diameter of coronary arteries from females was associated with elevated endothelial [Ca2+]i(female 174 nM, male 90 nM; P < 0.001). Elevation of Ca2+activated ecNOS with a similar slope and half-activation constant (∼160 nM) for both female and male coronary arteries. However, at [Ca2+] > 100 nM, ecNOS activity was significantly higher in coronary arteries from female rats compared with their male equivalents ( P < 0.01). Maximal activity for ecNOS at saturating Ca2+ (300 nM) was 37% higher in coronary arteries from female animals compared with male animals ( P < 0.05). Thus elevated [Ca2+]iin the endothelium of female coronary arteries alone is predicted to increase the production of NO (by nearly 2-fold). This gender difference combined with increased ecNOS activity at a given [Ca2+] in females indicates that tonic NO production should be nearly threefold greater in female coronary arteries compared with male coronary arteries. We conclude that, in the regulation of endothelial Ca2+ and ecNOS, gender differences contribute significantly to the overall decrease in myogenic tone observed in coronary arteries of females.