Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine-dependent growth of hematopoietic cells. (9/2958)

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin-3 (IL-3)-dependent cell line F-36P-mpl, the induced expression of dominant-negative (dn)-STAT5 and of dn-ras led to inhibition of IL-3-dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6-STAT5A) and ras (H-rasG12V) enabled F-36P-mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6-STAT5A and H-rasG12V individually and cooperatively transactivated the cyclin D1 promoter in luciferase assays. Both dn-STAT5 and dn-ras suppressed IL-3-induced cyclin D1 promoter activities in F-36P-mpl cells. Using a series of mutant cyclin D1 promoters, 1*6-STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT-binding sequence at -481 bp. In electrophoretic mobility shift assays, STAT5 bound to the element in response to IL-3. Furthermore, the inhibitory effect of dn-STAT5 on IL-3-dependent growth was restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine-dependent growth of hematopoietic cells.  (+info)

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae. (10/2958)

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.  (+info)

The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association. (11/2958)

Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition.  (+info)

Drosophila Src42A is a negative regulator of RTK signaling. (12/2958)

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.  (+info)

Rpb7 can interact with RNA polymerase II and support transcription during some stresses independently of Rpb4. (13/2958)

Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Delta, containing Pol IIDelta4) to grow above 30 degrees C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability of rpb4Delta cells to grow at 34 degrees C (a relatively mild temperature stress) but not at higher temperatures. Overexpression of RPB7 could also partially suppress the cold sensitivity of rpb4Delta strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Delta cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIDelta4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIDelta4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIDelta4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIDelta4, enough to permit appropriate transcription also under some stress conditions.  (+info)

SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle. (14/2958)

Recent work has demonstrated that the signal recognition particle (SRP) is required for the efficient insertion of many proteins into the Escherichia coli inner membrane (IM). Based on an analogy to eukaryotic SRP, it is likely that bacterial SRP binds to inner membrane proteins (IMPs) co-translationally and then targets them to protein transport channels ("translocons"). Here we present evidence that SecA, which has previously been shown to facilitate the export of proteins targeted in a post-translational fashion, is also required for the membrane insertion of proteins targeted by SRP. The introduction of SecA mutations into strains that have modest SRP deficiencies produced a synthetic lethal effect, suggesting that SecA and SRP might function in the same biochemical pathway. Consistent with this explanation, depletion of SecA by inactivating a temperature-sensitive amber suppressor in a secAam strain completely blocked the membrane insertion of AcrB, a protein that is targeted by SRP. In the absence of substantial SecA, pulse-labeled AcrB was retained in the cytoplasm even after a prolonged chase period and was eventually degraded. Although protein export was also severely impaired by SecA depletion, the observation that more than 20% of the OmpA molecules were translocated properly showed that translocons were still active. Taken together, these results imply that SecA plays a much broader role in the transport of proteins across the E. coli IM than has been previously recognized.  (+info)

Mutations in a dominant-negative isoform correlate with phenotype in inherited cardiac arrhythmias. (15/2958)

The long QT syndrome is characterized by prolonged cardiac repolarization and a high risk of sudden death. Mutations in the KCNQ1 gene, which encodes the cardiac KvLQT1 potassium ion (K+) channel, cause both the autosomal dominant Romano-Ward (RW) syndrome and the recessive Jervell and Lange-Nielsen (JLN) syndrome. JLN presents with cardiac arrhythmias and congenital deafness, and heterozygous carriers of JLN mutations exhibit a very mild cardiac phenotype. Despite the phenotypic differences between heterozygotes with RW and those with JLN mutations, both classes of variant protein fail to produce K+ currents in cultured cells. We have shown that an N-terminus-truncated KvLQT1 isoform endogenously expressed in the human heart exerts strong dominant-negative effects on the full-length KvLQT1 protein. Because RW and JLN mutations concern both truncated and full-length KvLQT1 isoforms, we investigated whether RW or JLN mutations would have different impacts on the dominant-negative properties of the truncated KvLQT1 splice variant. In a mammalian expression system, we found that JLN, but not RW, mutations suppress the dominant-negative effects of the truncated KvLQT1. Thus, in JLN heterozygous carriers, the full-length KvLQT1 protein encoded by the unaffected allele should not be subject to the negative influence of the mutated truncated isoform, leaving some cardiac K+ current available for repolarization. This is the first report of a genetic disease in which the impact of a mutation on a dominant-negative isoform correlates with the phenotype.  (+info)

Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process. (16/2958)

The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.  (+info)