UNIPROT:P67775 - FACTA Search

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Query: UNIPROT:P67775 (alpha isoform)
797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.
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PMID:Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A. 132 66

Regulation of Na,K-ATPase mRNA alpha isoform and mRNA beta expression by thyroid hormone (T3) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 abundances were nearly constant while mRNA alpha 2 was undetectable. During the interval between postnatal days 5 and 15, mRNA alpha 3 decreased to negligible levels and mRNA alpha 2 became expressed and increased in abundance to account for approximately 20% of the mRNA alpha pool by the 15th postnatal day. To examine the effect of T3 on this developmental program, neonates were injected with 75 micrograms T3/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T3 treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T3 treatment, the abundances of mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 increased, while mRNA alpha 2 continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T3 augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T3 does not act as a "molecular switch" in the developmental expression of the mRNA alpha isoforms in rat myocardium during the first four postnatal days.
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PMID:Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium. 164 35

Thyroid hormone receptors (TRs) are nuclear proteins that regulate gene expression through interactions with specific DNA sequences. It is well known that thyroid hormones have critical functions in the control of normal brain development. In the rat brain, at least three mRNA species are generated by differential processing of the TR alpha transcript. Only one of the isoforms, TR alpha-1, is a transcriptional activator, while the regulatory roles of the carboxy-terminal variants TR alpha-2 and TR alpha-2v remain unclear. In this study we have used polymerase chain reaction amplification of total RNA to compare TR alpha-1, TR alpha-2, and TR alpha-2v mRNA levels in the brainstem, cerebellum, cerebrum, midbrain, and olfactory bulbs of developing neonatal brains in rats. RNA was collected 5, 10, 15, 20, and 25 days after birth from both normal and hypothyroid animals. Coordinate expression of all three isoforms was observed in most tissues during development, with TR alpha-2 generally maintaining the highest level of expression, and TR alpha-1 the lowest. In hypothyroid tissues, TR alpha-1 message was generally increased, while TR alpha-2 was not. To explore the possible roles of the TR alpha isoforms, we have compared their DNA-binding activities. We report that compared to TR alpha-1, the carboxy-terminal variants TR alpha-2 and TR alpha-2v show different binding patterns with a thyroid hormone response element, suggesting that they bind only poorly as monomers. The varying ratios of the TR alpha isoform expression together with their distinct binding patterns and reported repressor functions suggest that TR alpha isoforms have important roles during brain development and function, and may serve to fine-tune the biological responses to thyroid hormone.
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PMID:Coordinate expression of functionally distinct thyroid hormone receptor alpha isoforms during neonatal brain development. 194 7

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.
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PMID:Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro. 254 40

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.
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PMID:Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A. 255 76

Heteroploid mouse NIH 3T3 fibroblasts and several rat fibroblast strains (Rat-1, Rat-2 and REF-52) are cell lines of special interest in the field of carcinogenesis because of their extensive use as normal cells in transformation assays for putative cancer-causing genes. Exposure of these cells to carcinogenic chemicals or oncogenic DNA produces anchorage-independent cells with retracted cytoplasms that lack actin cables. All human fibroblast strains, normal and transformed, synthesize two electrophoretic forms of actin (beta- and gamma-actin). In contrast, we discovered that early-passage mouse and rat strains synthesize abundant amounts of each of the three electrophoretic forms of actin (alpha-, beta- and gamma-actin) but mouse and rat cancer cells express only beta- and gamma-actins. We now show that in NIH 3T3 and Rat-2 fibroblasts a third actin, the smooth muscle alpha isoform, is abundantly co-expressed with beta- and gamma-actin. In every instance tested following transformation to tumorigenicity, the accumulation of alpha-actin messenger RNA and alpha-actin synthesis was greatly inhibited. Shutdown of alpha-actin expression thus appears to be a reproducible transformation-sensitive marker in rodent fibroblasts.
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PMID:Smooth muscle alpha-action is a transformation-sensitive marker for mouse NIH 3T3 and Rat-2 cells. 403 81

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.
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PMID:Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities. 756 77

HSF1 mediates the stress induced expression of heat shock proteins, referred to as the cellular stress response. Previous results indicated that mammalian cells express two distinct HSF1 protein isoforms, with molecular sizes of 69 kDa (HSF1-beta) and 71 kDa (HSF1-alpha). The purpose of this study was to determine the mechanism by which these two HSF1 protein isoforms are generated. Our results show that mammalian cells express two distinct HSF1 mRNA isoforms which arise via alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms differ by a single 66 bp exon of the HSF1 gene which is spliced into the HSF1-alpha mRNA isoform but skipped in the HSF1-beta mRNA isoform. This 66 bp exon encodes a 22 amino acid sequence, whose molecular weight (2.3 kDa) matches the difference in size between the HSF1-beta and HSF1-alpha protein isoforms (69 and 71 kDa). Further analysis reveals that this extra 22 amino acid sequence, whose insertion site in the HSF1-alpha isoform is located immediately adjacent to a C-terminal leucine zipper motif (leucine zipper 4) previously shown to be involved in maintenance of HSF1 in the non-DNA-binding control form, contains an additional, previously unidentified leucine zipper motif (leucine zipper 5). Our results also show that the levels of the two HSF1 isoforms are regulated in a tissue dependent manner, with testis expressing higher levels of the HSF1-beta isoform while heart and brain express higher levels of the HSF1-alpha isoform. These results demonstrate a new mechanism by which HSF1 expression is regulated in mammalian cells and suggest a potential role for the HSF1 isoforms in mediating tissue-dependent regulation of the cellular stress response.
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PMID:Regulated expression of heat shock factor 1 isoforms with distinct leucine zipper arrays via tissue-dependent alternative splicing. 759 26

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.
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PMID:The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice. 798 27

To characterize the expression of genes encoding the alpha- and beta-subunit isoforms of the Na(+)-K(+)-ATPase in rat kidney, we used reverse transcription (RT)-PCR of microdissected renal structures combined with quantitation of subunit isoform mRNAs in the major renal parenchymal zones. Transcripts for alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms were detected by RT-PCR in microdissected glomeruli, proximal convoluted tubules, medullary thick ascending limbs of Henle, cortical and inner medullary collecting ducts. The truncated alpha 1 (alpha 1-T) isoform was also amplified from cortex, outer and inner medulla and isolated glomeruli, but it was not detected in these nephron segments. The DNA sequence of the renal alpha 1-T PCR product was identical to that of the cDNA previously cloned from aortic smooth muscle cells. RNA dot-blot analysis indicated that the alpha 1, alpha 2, and alpha 3 isoforms contributed approximately 70%, approximately 20%, and approximately 10%, respectively, of the total alpha isoform mRNA in each parenchymal zone. RNase protection assays determined that the beta 1 and beta 2 isoforms accounted for approximately 95% and approximately 5%, respectively, of the beta isoform mRNA in each zone. These data provide definitive evidence for the differential expression of mRNAs encoding all the alpha and beta isoforms in the renal parenchyma, and for the coexpression of these isoforms in the nephron segments examined. The results suggest the potential expression of up to eight different Na(+)-K(+)-ATPase isoenzymes in the kidney, and for multiple molecular levels of regulation of renal Na(+)-K(+)-ATPase expression.
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PMID:Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha- and beta-subunit isoforms in rat kidney using RT-PCR. 799 86

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