Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

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.

The polypeptide chain of eukaryotic initiation factor 5A occurs in two distinct conformations in the absence of the hypusine modification.

Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity. We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride. In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2.

Identification of a new member of the human eIF-5A gene family.

Using an oligodeoxynucleotide generated by rapid PCR amplification of 5'-cDNA ends (5'-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A). Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns. In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A.

Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes.

Distinct sequence motifs within the cytoplasmic domain of the human IL-4 receptor differentially regulate apoptosis inhibition and cell growth.

Hematopoietin receptors generally function as multimeric complexes composed of a unique ligand-binding chain and a second component often shared between several members of this receptor family. To better understand the signal transduction of the human IL-4 receptor (hIL4R), we analyzed the functionality of targeted mutations in two cytoplasmic regions of the ligand-binding hIL4R chain that we previously identified to be necessary for growth mediation in factor-dependent murine Ba/F3 cells. Here, we provide evidence that transient inhibition of apoptotic death of Ba/F3 cells and the competence to proliferate indefinitely depend on separated and distinct sequence motifs of the hIL4R.