Gene-hypermutated isoforms of MYC protein: Tumour-suppressor P-53 protective-initiation of apoptosis that normally prevents Burkitt's lymphoma tumorigenesis can be enhanced.
Explore the source record for details and available documents.
Animals↗
SEARCH · PubMed Health
Results for “Protein Isoforms”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Regulation of CCAAT/enhancer-binding protein-beta isoform synthesis by alternative translational initiation at multiple AUG start sites.
The mRNA of the intronless, single-copy CCAAT/enhancer-binding protein-beta (C/EBPbeta) gene encodes several isoforms that have truncated transcription activation domains. This occurs by the alternative translational initiation (ATI) at multiple AUG start sites. The C/EBPbeta mRNA has four in-frame AUGs and an internal out-of-frame AUG associated with a small open reading frame (sORF). Initiation of translation at the in-frame AUGs forms 40-kDa (AUG-1), 35-kDa (AUG-2), 20-kDa (AUG-3) and 8.5-kDa (AUG-4) isoforms. We show that in COS-1 cells the 20-kDa isoform is not a product of proteolysis of the higher molecular weight isoforms. The sORF contains an AUG and termination signal that may produce the oligopeptide MPPAAARRL. Our studies suggest that ATI involves three mRNA structural features: (i) the cap structure, (ii) the context of the Kozak sequences that flank the AUG and (iii) the integrity of the sORF. We propose that formation of C/EBPbeta isoforms is accomplished by a leaky ribosomal scanning mechanism that facilitates ATI of multiple internal AUGs.
Alternative Splicing↗
Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins.
Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.
Amino Acid Sequence↗
Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance.
Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-kappaB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRalpha), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRbeta), which does not bind known ligands and attenuates GRalpha action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-kappaB DNA binding site 5' to the hGR promoter that leads to a 1.5-fold increase in GRalpha mRNA and a 2.0-fold increase in GRbeta mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-alpha treatment disproportionately increased the steady-state levels of the GRbeta protein isoform over GRalpha, making GRbeta the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-alpha or IL-1. The increase in GRbeta protein expression correlated with the development of glucocorticoid resistance.
Animals↗
Distinct isoforms of protein kinase C are involved in human eosinophil functions induced by platelet-activating factor.
BACKGROUND: Platelet-activating factor (PAF) is a potent stimulator of eosinophils. Recently, treatment with a protein kinase C (PKC) inhibitor which generally inhibits PKC isoforms has been shown to modulate several eosinophil functions in distinct manners, in that PKC inhibition enhanced CD11b expression and cellular adhesion, but inhibited superoxide generation and degranulation in PAF-stimulated human eosinophils. These results suggested that distinct PKC isoforms were likely to be involved in each eosinophil function induced by PAF. We have therefore investigated whether or not the PKC isoforms involved in PAF-induced CD11b expression and superoxide generation were different. METHODS: Human eosinophils prepared from healthy volunteers were treated with PKC inhibitors, bis-indolylmaleimide I (BisI; a general PKC inhibitor), myristoylated PKC inhibitor peptide (myr-psiPKC; a PKCalpha, beta and delta inhibitor) and rottlerin (a PKCdelta inhibitor), followed by stimulation with PAF. CD11b expression was determined using flow cytometry and superoxide generation was evaluated using a cytochrome c reduction assay. RESULTS: BisI treatment led to enhancement of PAF-induced CD11b expression, while myr-psiPKC and rottlerin did not. In contrast, PAF-induced superoxide generation was inhibited by treatment with BisI, myr-psiPKC and rottlerin. CONCLUSIONS: PKCalpha, beta and delta are not involved in PAF-induced CD11b expression, but PKCdelta is involved in the PAF-induced activation of superoxide anion generation.
Acetophenones↗
Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution.
Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aalpha and Abeta. Mutations have been found in both isoforms in a variety of human cancers. Although Aalpha has been intensely studied, little is known about Abeta. We generated Abeta-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Abeta in comparison with Aalpha. Both forms were expressed at constant levels throughout the cell cycle, but Aalpha was expressed at a much higher level than Abeta. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10 h. However, Aalpha and Abeta differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aalpha was expressed at similarly high levels in all tissues and cell lines, Abeta expression varied greatly. In addition, in vivo studies with epitope-tagged Aalpha and Abeta subunits demonstrated that Abeta is a markedly weaker binder of regulatory B and catalytic C subunits than Aalpha. Construction of phylogenetic trees revealed that the conservation of Aalpha during the evolution of mammals is extraordinarily high in comparison with both Abeta and cytochrome c, suggesting that Aalpha is involved in more protein-protein interactions than Abeta. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aalpha and Abeta. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aalpha bound SV40 small tumour antigen.
Animals↗
Mammalian skeletal muscle fiber type transitions.
Mammalian skeletal muscle is an extremely heterogeneous tissue, composed of a large variety of fiber types. These fibers, however, are not fixed units but represent highly versatile entities capable of responding to altered functional demands and a variety of signals by changing their phenotypic profiles. This adaptive responsiveness is the basis of fiber type transitions. The fiber population of a given muscle is in a dynamic state, constantly adjusting to the current conditions. The full range of adaptive ability spans fast to slow characteristics. However, it is now clear that fiber type transitions do not proceed in immediate jumps from one extreme to the other, but occur in a graded and orderly sequential manner. At the molecular level, the best examples of these stepwise transitions are myofibrillar protein isoform exchanges. For the myosin heavy chain, this entails a sequence going from the fastest (MHCIIb) to the slowest (MHCI) isoform, and vice-versa. Depending on the basal protein isoform profile and hence the position within the fast-slow spectrum, the adaptive ranges of different fibers vary. A simple transition scheme has emerged from the multitude of data collected on fiber type conversions under a variety of conditions.
Animals↗
Regulators of G-protein signaling form a quaternary complex with the agonist, receptor, and G-protein. A novel explanation for the acceleration of signaling activation kinetics.
Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the Galpha subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K+ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein alpha subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein alpha subunit.
Cell Line↗
Differential tyrosyl-phosphorylation of multiple mitogen-activated protein kinase isoforms in response to prolactin in Nb2 lymphoma cells.
Prolactin (PRL) stimulates mitogenesis and differentiative processes in a variety of cell types. Not all of the molecules involved in PRL signaling, which follows an initial PRL-receptor interaction, have been identified. In the present studies, PRL is shown to stimulate the differential tyrosyl phosphorylation of three isoforms (ERK-1, 2, and 4) of mitogen-activated protein kinases (MAP kinase) in a rat pre-T lymphoma cell line (Nb2). Evidence also suggests that PRL stimulates the tyrosyl phosphorylation of ERK-3, a MAP kinase isoform recently identified. When G1-arrested Nb2 cells are treated with 50 ng/ml oPRL, ERK-1 through 3 become tyrosyl phosphorylated within minutes (an indication of enzyme activation) and then become dephosphorylated within 30 min. Conversely, ERK-4 is rapidly tyrosyl phosphorylated by 5 min, and remains in this state for at least 1 hr.
Animals↗
Identification of three isoform patterns of human serum amyloid A protein.
Three patterns of human apo-SAA (serum amyloid A protein) isoforms have been identified by electrofocusing. In pattern 1, six major apo-SAA isoforms of pI 6.0, 6.4, 7.0, 7.4, 7.5 and 8.0 were found. In pattern 2, the apo-SAA isoforms of pI 7.4 and 8.0 were not detected, whereas in pattern 3 the pI-7.0 and -7.5 isoforms were lacking. Six patients displayed apo-SAA isoform pattern 1, 11 displayed pattern 2 and one displayed pattern 3.
Adult↗
Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms.
DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.
Alternative Splicing↗
Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength.
We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.
Amino Acid Sequence↗
Developmental expression of the fragile X-related 1 proteins in mouse testis: association with microtubule elements.
Fragile X mental retardation 1 protein (FMRP) is the archetype of a class of cytoplasmic mRNA-binding proteins that includes the fragile X-related 1 and 2 proteins (FXR1P and FXR2P). Whereas absence of FMRP is the cause of fragile X syndrome, it is not known if FXR1P and FXR2P are associated with any pathology. It is also still elusive whether these homologous proteins can partially compensate for the absence of FMRP in the case of the fragile X syndrome. FXR1 is widely expressed in mammals and its expression pattern is complex since several mRNA variants and protein isoforms are detected. In mouse, we observed that the highest level of FXR1 is found in the adult testis. This tissue is an exception, since all known FXR1P isoforms, some of which have been considered as tissue specific, are detected in it. In young animals, changes in mRNA-spliced variants and their corresponding protein isoforms occur during spermatogenesis. Using biochemical, immunohistochemical and electron microscopic techniques, we show that FXR1P is associated with microtubule elements. Since the cytoskeletal framework is implicated in cellular plasticity as well as in mRNA transport, we propose new possibilities for the function(s) of the FXR proteins.
Aging↗
Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.
1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U-purinoceptors. Both downregulation and inhibition studies show that PKC-alpha is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-epsilon (PKC-zeta is unresponsive to PMA), or an as yet uncharacterized PKC isoform.
6-Ketoprostaglandin F1 alpha↗
[Is alternative splicing of mammalian genes conservative?].
The conservation of alternative splicing in orthologous genes from the human and mouse genomes was analyzed. Alternatively spliced mouse genes from the AsMamDB database were used to scan the draft human genome. The mouse protein isoforms were aligned with respect to orthologous human genes, and thus the exon-intron structure of the latter was established. Proteins isoforms that could not be aligned throughout their length were analyzed in detail using the human EST alignment.
Alternative Splicing↗
Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure.
To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.
Adipose Tissue, Brown↗
Isoforms of protein kinase C in postsynaptic densities after cerebral ischemia.
Relatively mild ischemic insult can lead to delayed neuronal cell death in vulnerable brain regions. We provide evidence that the protein composition of the postsynaptic densities (PSD) undergoes rapid modification after 15 min postdecapitative as well as 5 min transient global ischemia. We observed a significant increase in cPKC and nPKC protein content in the postischemic PSD. Of the calcium-regulated PKC isoforms, the alpha and beta subtypes increase in PSD over ten times above the control values whereas gamma PKC, an isoform most abundant in the native PSD structure, shows relatively smaller changes under ischemic conditions. For the first time, the PSD membrane translocation of Ca(2+)-independent isoforms delta and epsilon is shown. The yield of the PSD protein preparation from the postischemic cortex was two times higher compared with control. This correlated with an abundant increase in electron density and changes in ultrastructure of PSD isolated from postischemic cortex. Also sections from CA1 gerbils hippocampus after transient ischemia showed persistent enlargement of postsynaptic densities up to 24 h of reperfusion. This was accompanied by elevation of the PSD/cytoskeleton-associated alpha, beta PKC immunoreactivity and other changes in neuronal and glial cell morphology typical of the early postischemic degeneration. Sustained changes in PKC composition and organization of postsynaptic membranes during and after ischemia may cause persistent alteration in synaptic transmission and subsequently contribute to delayed neuronal injury.
Animals↗
Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation.
Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.
Animals↗