Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen–mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc∈ receptor I by modulating the balance of the counterregulatory lipids.

Cap 'n' collar-basic leucine zipper (CNC-bZIP) proteins are widely implicated in developmental processes throughout different species. Evidence is accumulating that some of them are also participating in induced gene expression in the adult. Here we show that the three CNC-bZIP members NF-E2, Nrf1 and Nrf2 are constitutively expressed in the murine mast cell line CPII and that they form transcription factor complexes with several AP1 binding proteins. Upon induction, complexes are observed at the 2 x NF-E2 consensus binding site and the extended kappa3/AP1(+) site of the TNFalpha promoter. The interaction of Nrf1 with c -jun, junD, fosB and ATF2 in mast cells is in contrast to the recently reported binding of Nrf1 alone at the kappa3/AP1(-) site in dendritic cells. We speculated that this may be the result of the expression of isoforms of Nrf1 in mast cells. Using a PCR cloning strategy, we have isolated six novel splice variants of this transcription factor. Some of them have deleted the translational stop codon, resulting in an Nrf1 protein with an altered leucine zipper region. Expression of this altered binding/interaction domain interferes with TNFalpha induction, indicating an interaction of this splice variant with the active AP1/NF-AT complex at this promoter.

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca 2؉ , released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-␥ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcRI triggering, enhanced sphingosine kinase activity was associated with FcRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn ؊/؊ mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcRI proximal event.In addition to the importance of sphingolipids in membrane structure, the sphingomyelin pathway with its lipid products ceramide, sphingosine (S), and S-1-phosphate (S1P) has been recognized to function in a variety of signaling events (3, 36). Many of the sphingolipids generated along this pathway are secreted and bind specific cellular receptors on a variety of cell types. On the other hand, they also function intracellularly as "second messenger molecules" similar to the various intermediates of phosphoglyceride metabolism (3, 36). Processes such as apoptosis, differentiation, and cell activation are directly regulated or fine-tuned by ceramide and its derivatives. However, in contrast to glycerolipids, the individual concentrations of various intracellular sphingolipids serve to balance cell activation and inactivation. This concept has been termed the sphingolipid rheostat (3). Specific examples include ceramide and S1P, whose relative intracellular concentration regulates apoptosis; S and S1P, which regulates immunoglobulin E-antigen (IgE-Ag) sensitivity in mast cells; and ceramide and ceramide-1-phosphate, which regulates the process of phagocytosis in macrophages (2,14,29,37,42,43). An essential cellular checkpoint in the sphingomyelin signaling pathway is S kinase (SPHK), as its product S1P is able to counteract ceramide as well as S-mediated effects in apoptosis and effector function regulation (e.g., inhibition of cytokine induction by S in mast cells). In humans, two different isoforms and several splice variants of SPHK were described and cloned (34). While activation of SPHK1 has been firmly established (e.g., after FcεRI and Fc␥RI signaling), its relationship to other known mast cell signaling molecules is still unexplored (24, 37). Recent data on Fc␥RI positioned SPHK downstream of phospholipase D, with a de...

Posttranslational modification of a specific lysine residue in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability and proliferation. The product of this modification is hypusine, an amino acid unique to eIF-5A. We have purified and characterized one major and three minor isoforms of human eIF-5A from HeLa cells. The main form, which accounts for approximately 95% of the total eIF-5A, carries hypusine at position 50 and is amino-terminally acetylated as determined by amino acid composition analysis and electrospray ionization mass spectrometry. Analytical gel filtration indicates that this protein variant possesses a native apparent molecular weight that lies between that expected for a monomeric and dimeric form. Nevertheless, several experiments confirm this protein to be monomeric. It is further shown that eIF-5A have well-defined secondary structure. Both the far-UV circular dichroism spectrum as well as secondary structure predictions using different algorithms suggest this protein to have predominantly beta-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to the main form, all three minor isoforms of eIF-5A are characterized by acetylation of the epsilon-amino group of lysine at position 47. The minor isoforms are distinguishable by their state of modification of the lysine residue at position 50. Whereas the main form occurs in both the cytoplasmic and the nuclear fraction of HeLa cells, the minor isoforms were not detectable in the preparation of the nuclear fraction. Therefore, acetylation of lysine at position 47 might play a controlling role in the distribution of the minor isoforms to the nucleus.