Temperature-sensitive transforming mutants of the v-rel oncogene.

By making site-directed mutations in the avian retroviral oncogene v-rel, we created two temperature-sensitive (ts) transforming mutants; these changes were analogous to mutations previously shown to confer a ts function onto the Dorsal protein of Drosophila melanogaster. Chicken spleen cells infected with the ts v-rel mutants formed colonies in agar at 36.5 degrees C but not at 41.5 degrees C. In addition, spleen cells derived from the ts v-rel-transformed colonies could be propagated in liquid culture at 36.5 degrees C but rapidly senesced at 41.5 degrees C. Both mutant v-Rel proteins were also ts for DNA binding in vitro. These mutants may be valuable for identifying genes directly regulated by v-rel.     

Functional insights into the activation mechanism of Ste20-related kinases

Mammalian Ste20-related kinases modulate salt transport and ion homeostasis through physical interaction and phosphorylation of cation-chloride cotransporters. Identification of a sea urchin (Strongylocentrotus purpuratus) ortholog of the mouse Oxidative Stress Response 1 (OSR1) kinase prompted the cloning and testing of the functional effect of a non-mammalian kinase on a mammalian cotransporter. Heterologous expression of sea urchin OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevisoocytes activated the cotransporter indicating evolutionary conservation of the WNK4-OSR1-NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full-length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras by replacing specific portions of the suOSR1 gene with complimentary mouse OSR1 sequences. Co-expression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which possibly prevented the kinase from acquiring an active conformation. Interestingly, non-functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co-expressed in frog oocytes. Sea urchin/mouse OSR1 chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20-related kinases.     

Structural insights into the molecular mechanism of H‐NOX activation

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron–histidine (Fe–His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H‐NOX (Heme‐Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H‐NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H‐NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high‐resolution crystal structure of an H‐NOX mutant mimicking a broken Fe–His bond is reported. This mutant exhibits specific changes in heme conformation and major N‐terminal displacements relative to the wild‐type H‐NOX protein. Fe–His ligation is ubiquitous in all H‐NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H‐NOX family when NO binding leads to rupture of the Fe–His bond.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

Zinc finger proteins: insights into the transcriptional and post transcriptional regulation of immune response

Molecular Biology Reports - Zinc finger proteins encompass one of the unique and large families of proteins with diversified biological functions in the human body. These proteins are primarily...