The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs).
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Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor. (+info)
Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein.
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The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase. (+info)
BACKGROUND: Obstructive nephropathy leads to progressive renal tubular atrophy and interstitial fibrosis and is associated with sodium wasting and sodium depletion. Renal damage resulting from unilateral ureteral obstruction (UUO) may be aggravated by reactive oxygen species (ROS), which are produced by a variety of processes. Ideally, deleterious effects of ROS are attenuated by antioxidant enzymes, including the superoxide dismutases, glutathione peroxidases, catalase, and glutathione-S-transferases. The general paradigm is that tissue damage occurs when ROS production is greater than the protective capacity of the antioxidant enzymes. METHODS: This study was designed to investigate the response of renal antioxidant enzymes to UUO and sodium depletion. Adult, male Sprague-Dawley rats received normal-sodium or sodium-depleted siets and were subjected to UUO or sham operation. Obstructed (UUO), intact opposite, or sham-operated kidneys were harvested after 14 days, and antioxidant enzyme activities were measured in kidney homogenates. Thiobarbituric acid reactive substances were measured in these homogenates at 3 and 14 days after UUO or sham operation as an index of ROS production. RESULTS: Renal interstitial area, a measure of fibrosis, was increased by UUO and was doubled in sodium-depleted animals. Sodium depletion increased manganese superoxide dismutase, glutathione peroxidases, and glutathione-S-transferase activities in sham-operated kidneys but not in UUO kidneys. Relative to intact opposite kidneys, UUO kidneys had reduced activities of catalase, manganese superoxide dismutase, and glutathione-S-transferase in normal-sodium animals and all antioxidant enzymes tested in sodium-depleted animals. Renal thiobarbituric acid reactive substances were increased by three days of UUO and were increased further by 14 days of sodium depletion. CONCLUSION: In summary, sodium depletion increased several renal antioxidant enzymes, consistent with a stress response to increased ROS production. Further, UUO not only reduced antioxidant enzyme activities but also inhibited increases seen with sodium depletion. We conclude that suppression of renal antioxidant enzyme activities by UUO contributes to the progression of renal injury in obstructive nephropathy, a process exacerbated by sodium depletion. (+info)
Expression of disulphide-bridge-dependent conformational epitopes and immunogenicity of the carboxy-terminal 19 kDa domain of Plasmodium yoelii merozoite surface protein-1 in live attenuated Salmonella vaccine strains.
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The 19 kDa carboxy-terminal domain of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) was expressed in Salmonella vaccine strains as a carboxy-terminal fusion to fragment C of tetanus toxin (TetC). This study demonstrates that antibodies that recognize disulphide-dependent conformational epitopes in native MSP1 react with the TetC-MSP1(19) fusion protein expressed in Salmonella. The proper folding of MSP1(19) polypeptide is dependent on both the Salmonella host strain and the protein to which the MSP1(19) polypeptide is fused. Serum from mice immunized with Salmonella typhimurium C5aroD expressing TetC-MSP1(19) recognized native MSP1 as shown by immunofluorescence with P. yoelii-infected erythrocytes. Antibody levels to MSP1(19) were highest in out-bred mice immunized with S. typhimurium C5aroD carrying pTECH2-MSP1(19) and antibody was mostly directed against reduction-sensitive conformational epitopes. However, antibody levels were lower than in BALB/c mice immunized with a glutathione S-transferase (GST)-MSP1(19) fusion protein in Freund's adjuvant, and which were protected against P. yoelii challenge infection. In challenge experiments with P. yoelii the Salmonella-immunized mice were not protected, probably reflecting the magnitude of the antibody response. The results of this study have important implications in the design of live multivalent bacterial vaccines against eukaryotic pathogens. (+info)
Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.
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It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development. (+info)
Panning for genes--A visual strategy for identifying novel gene orthologs and paralogs.
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We have developed a rapid visual method for identifying novel members of gene families. Starting with an evolutionary tree, 20-50 protein query sequences for a gene family are selected from different branches of the tree. These query sequences are used to search the GenBank and expressed sequence tag (EST) DNA databases and their nightly updates using the tfastx3 or tfasty3 programs. The results of all 20-50 searches are collated and resorted to highlight EST or genomic sequences that share significant similarity with the query sequences. The statistical significance of each DNA/protein alignment is plotted, highlighting the portion of the query sequence that is present in the database sequence and the percent identity in the aligned region. The collated results for database sequences are linked using the WWW to the underlying scores and alignments; these links can also be used to perform additional searches to characterize the novel sequence further. With traditional "deep" scoring matrices (BLOSUM50) one can search for previously unrecognized families of large protein superfamilies. Alternatively, by using query sequences and EST libraries from the same species (e. g., human or mouse) together with "shallow" scoring matrices and filters that remove high-identity sequences, one can highlight new paralogs of previously described subfamilies. Using query sequences from the glutathione transferase superfamily, we identified two novel mammalian glutathione transferase families that were recognized previously only in plants. Using query sequences from known mammalian glutathione transferase subfamilies, we identified new candidate paralogs from the mouse class-mu, class-pi, and class-theta families. (+info)
Glutathione S-transferase-mu (GSTM1) and -theta (GSTT1) genotypes in the etiology of prostate cancer.
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The glutathione S-transferases (GSTs) are involved in the metabolism of numerous potential prostate carcinogens. Common homozygous germ-line deletions exist in the genes that encode GST-mu (GSTM1) and GST-theta (GSTT1) and preclude enzyme expression. To evaluate whether GSTM1 and/or GSTT1 contribute to prostate cancer (CaP) etiology, we studied 237 incident CaP cases and 239 age- and race-matched controls. The probability of having CaP was increased in men who had nondeleted (functional) genotypes at GSTT1 (odds ratio, 1.83; 95% confidence interval, 1.19-2.80) but not GSTM1 (odds ratio, 1.07; 95% confidence interval, 0.74-1.55). No interaction of these genes in CaP etiology was observed. GST-theta is highly expressed in the prostate and can produce genotoxic effects upon exposure to specific carcinogens. These results suggest that GSTT1 is associated with CaP risk. (+info)
Polymorphisms in GSTP1, GSTM1, and GSTT1 and susceptibility to colorectal cancer.
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Polymorphisms in glutathione S-transferase (GSTs) may predispose to colorectal cancer through deficient detoxification of environmental carcinogens, although previous results are conflicting. A study with 178 matched case-control pairs was conducted to determine the effect of the GSTT1 and GSTM1 null genotypes and polymorphisms in GSTP1 on colorectal cancer susceptibility. In a secondary analysis, we examined interactions between genotypes and with the N-acetyltransferase 2 (NAT2) genotype. Heterogeneity by age, sex, site, and stage of cancer was also examined. No effect of any genotype for GSTM1, GSTT1, or GSTP1 on colorectal cancer susceptibility was detected. Secondary end points showed that individuals with both the GSTT1 null and NAT2 slow genotypes combined appeared to be at increased risk of colorectal cancer (odds ratio = 2.33; 95% confidence interval, 1.1-5.0). We conclude that GST polymorphisms alone do not predispose to colorectal cancer in northeast England. We also observed possible effects of the GSTT1 null genotype on the age and stage at presentation, and these, together with the findings of an apparent interaction with NAT2 genotypes, need to be confirmed in further studies. (+info)