The human immunodeficiency virus type 1 (HIV-1) transactivator Rev is a nuclear protein that regulates expression of certain HIV-1 transcripts by binding to an RNA target element (the RRIE) present in these transcripts. A short arginine-rich sequence in Rev contains the signals required to direct this protein into nuclei, where it associates preferentially with nucleoli. We created a steroid-inducible transactivator by fusing Rev with the steroid-binding domain of the glucocorticoid receptor (GR). This Rev/GR protein remains inactive in the cytoplasm when steroids are absent, but it enters the nucleus and initiates transactivatlon within minutes after exposure to dexamethasone. Although the GR moiety is sufficient to transport Rev/GR into nuclei, mutation of certain residues in the arginine-rich region blocks nucleolar localization and also inhibits transactivation. We find that other mutations in this region, however, can abolish the function of Rev/GR without affecting its localization; the latter phenotype may reflect a specific defect in binding of the RRE.The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116-amino acid regulatory protein that transactivates the expression of other essential HIV-1 genes (1, 2). Localized in the nuclei of infected cells, where it associates preferentially with nucleoli (3), Rev acts at the posttranscriptional level by inducing the export of incompletely spliced viral mRNAs to the cytoplasm (1, 2, 4-8). The mechanism ofthis transactivation is not fully understood, but it is thought to depend on the binding of Rev protein to an array of RNA hairpin loops (termed the Rev response element, RRE) situated within the target HIV-1 transcripts (9)(10)(11)(12)(13)(14).The signals that direct Rev protein into nuclei are contained in a short basic region of the protein (approximately amino acids 25-50) that is rich in arginine residues (15, 16). Mutations in this basic region inhibit the nuclear accumulation of Rev and produce a concomitant defect in transactivation, implying that Rev must enter the nucleus to exert its effects. Although recent reports suggest that this region also harbors sequences that target Rev to nucleoli (17, 18) or participate in binding of the RRE (19), it has been difficult to evaluate these activities by conventional mutagenic analysis in vivo because of the need to maintain efficient nuclear translocation. We now describe studies of a Rev fusion protein whose entry into the nucleus is controlled by sequences from the glucocorticoid receptor (GR). This Rev/GR protein exhibits steroid-dependent Rev activity and thus provides the basis for an inducible system in which to study the dynamics of Rev transactivation. Moreover, because the GR moiety is sufficient to direct this protein into nuclei, mutational analysis ofRev/GR has enabled us to evaluate the functions of the basic region independently of any effect on nuclear translocation. Our findings illuminate the critical and complex role of this region in the localization and function ...
The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R., Taagepera
The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. Previously, we identified a human Cdc7 homolog, HsCdc7. In this study, we report the identification of a human Dbf4 homolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells.
Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393-407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half-maximal binding of 125I-SPR393-407 of 1:70,000. Nonradioactive SPR393-407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393-407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I-substance P (apparent Kd of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393-407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393-407 or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393-407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393-407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.
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