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Results for “Protein Isoforms”

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PubMed1989

Developmental and muscle-specific changes in methylation of the myosin light chain LC1f and LC3f promoters during avian myogenesis.

The fast alkali myosin light chains LC1f and LC3f are two contractile protein isoforms encoded for by a single gene complex. Expression of these two isoforms is dependent upon differential initiation of transcription at either of two promoters encoding unique 5' exons for isoform-specific amino termini of these light chains. Studies of protein expression have shown that the two isoforms are first expressed at different stages of development and in the case of the LC3f isoform only in fast twitch muscle fiber types. The molecular mechanisms that regulate the differential transcription of the gene complex are unknown. Experiments reported here demonstrated the direct correlation of isoform protein and mRNA expression with the undermethylation of the DNA in the promoter regions of the gene for each of the expressed myosin light chain isoforms. We find that fast and slow muscles have different patterns of undermethylation of the two promoter regions of the gene. Moreover, changes in methylation of the promoter regions were shown to occur specifically in skeletal muscle tissue, to be developmentally regulated, and to only occur in the LC3f promoter of those muscle groups that express LC3f protein.

Age Factors↗

PubMed1998

Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3'-kinase activity.

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3'-kinase (PI 3'-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by alphaIRS-1(mostly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alphaShc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, alphaShc immunoprecipitated only the p85 isoform; the associated PI 3'-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (alphaIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Improved separation of microheterogeneities and isoforms of proteins by capillary electrophoresis using segmental filling with SDS and PEO in the background electrolyte.

To improve the separation efficiency while achieving high sensitivity for the analysis of proteins' microheterogeneity, a segmental-filling technique has been developed and tested in capillary electrophoresis with laser-induced native fluorescence using a pulsed Nd:YAG laser. Using a short plug of SDS applied to the capillary and the anticonvectant poly(ethylene oxide) (PEO), the microheterogeneities of a number of proteins with pI values ranging from 4.5 to 11.1 could be detected. This high resolving power is due to reduced adsorption on the capillary wall, sieving, and the interaction with SDS. Consequently, the length and the concentration of the SDS plug play a significant role in determining the resolution and sensitivity. The method has been applied to the analysis of salivary and cerebrospinal fluid (CSF) samples. Without any sample pretreatment, using a 10-s 1x SDS plug, six alpha-amylase isoforms in a salivary sample were resolved in 17 min and three more peaks were detected in a CSF sample. With simplicity, high resolving power, and rapidity, the method has shown great potential for proteomics.

Electrolytes↗

PubMed2002

Analysis of the human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mechanism.

We determined the gene structure of the human TrkB gene. The gene is unusually large and spans at least 590 kbp. It contains 24 exons. Using alternative promoters, splicing, and polyadenylation sites, the gene can create at least 100 isoforms, that can encode 10 proteins. RT-PCR and Northern blot analysis reveals that only three major protein isoforms are generated by the gene: the full length receptor, an isoform lacking the tyrosine kinase domain, and a novel isoform lacking the tyrosine kinase domain but containing a Shc binding site. This novel isoform, TrkB-T-Shc is generated by the use of a new alternative exon 19. It is expressed only in brain. TrkB-T-Shc protein is located in the plasma membrane. Coimmunoprecipitation experiments show that TrkB-T-Shc is not phosphorylated by the full length receptor, indicating that it could be a negative regulator of TrkB signaling in the brain.

5' Untranslated Regions↗

PubMed2006

A specific catalytic subunit isoform of protein kinase CK2 is required for phosphorylation of the repressor Nrg1 in Saccharomyces cerevisiae.

Protein kinase CK2 is highly conserved in eukaryotes and plays roles in many different cellular processes. CK2 is a tetramer comprising two catalytic and two regulatory subunits. Most organisms have two major isoforms of the catalytic subunit, and evidence suggests strongly overlapping function. In the yeast Saccharomyces cerevisiae, CK2 is essential for viability, and either catalytic subunit isoform, Cka1 or Cka2, suffices, but previous genetic evidence suggests that the isoforms have some distinct roles. In this work, we present evidence that the transcriptional repressor Nrg1, which regulates various stress-responsive genes, is a downstream target of CK2 containing the Cka1 isoform. We found that Nrg1 is phosphorylated in response to stress and that its phosphorylation was defective in cka1Delta, but not cka2Delta, mutants. Thus, the Cka1 catalytic subunit isoform is specifically required for phosphorylation of Nrg1 in vivo. The CK2 regulatory subunits were also required, indicating that the CK2 holoenzyme is involved. Both yeast and recombinant human CK2 phosphorylated recombinant Nrg1 in vitro. This identification of a protein whose phosphorylation requires a specific CK2 catalytic subunit isoform supports the view that the two isoforms exhibit functional specificity in vivo.

Casein Kinase II↗

PubMed2000

Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase.

Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.

Animals↗

PubMed2003

Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques.

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.

Amino Acid Sequence↗

PubMed2000

Protein kinase cdelta and alpha are involved in the development of vasospasm after subarachnoid hemorrhage.

We have previously shown the enhanced activity of protein kinase C in the membrane fraction of the canine vasospastic artery after subarachnoid hemorrhage, which increased with progression of angiographic vasospasm. This study examined identification of protein kinase C isoforms in the canine basilar artery, and the changes in expression and/or translocation of each isoform during the development of vasospasm. Vasospasm was produced by using the "two-hemorrhage" canine model in the basilar artery, and angiographic progression of vasospasm was assessed consecutively. Two isoforms, protein kinase Calpha and delta were identified in basilar arteries by Western blotting. Densitometric analysis showed that the expression of protein kinase Cdelta in the membrane fraction was significantly increased in the earlier stage, and protein kinase Calpha was increased later as vasospasm progressed. These results indicate that protein kinase Cdelta and alpha isoforms may play a significant role in the development and maintenance of vasospasm.

Actins↗

PubMed2004

No ING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma.

The ING1 family of proteins has been shown to have regulatory functions in oncogenesis, apoptosis, DNA repair and cell cycle regulation. Here we present the first report on LOH analysis of the ING1 locus, mutation analysis of the complete coding sequence including intron-exon boundaries and expression analysis of the different ING1 splice products and protein isoforms in primary brain tumours. No somatic ING1 mutations were detected. Semi-quantitative analysis revealed higher levels of p33ING1b RNA in benign than in malignant lesions. This correlation was significant in a subset of 37 astrocytic tumours WHO grades I to IV. ING1 protein isoforms p47ING1a, p33ING1b and p24ING1c were found to be expressed variably in this series. Our findings support a regulatory contribution of ING1 to the development or progression of brain tumours.

Astrocytoma↗

PubMed1997

Developmental testis-specific regulation of mRNA levels and mRNA translational efficiencies for TATA-binding protein mRNA isoforms.

Early spermatids contain roughly 1000-fold more TATA-binding protein (TBP) mRNA than do somatic cells. The appearance of TBP-overexpressing spermatids in the developing testis is accompanied by a large increase in whole-organ levels of total RNA and of poly(A)+ RNA per cell. Whereas somatic cells initiate transcription of TBP mRNA at a single promoter/first exon (exon 1C), in adult testis, two additional major promoter/first exons (1D and 1E) are used. We have examined the expression of the somatic and testis-specific TBP mRNA isoforms during rodent testis development. In juvenile testes TBP mRNAs containing either exon 1C or exon 1D, but none containing exon 1E, are detected. At 21 days of age, all TBP mRNA isoforms begin to overaccumulate. The onset of TBP mRNA overaccumulation is marked first by an increase in levels of polysomal TBP mRNA, and later by accumulation of mRNP-associated TBP mRNA. In adult testes, only 30% of the total TBP mRNA is engaged by polysomes; the remainder is sequestered as mRNP particles. All of the TBP mRNA isoforms in adults exist both as free mRNP particles and as polysomes; however, the fraction in polysomes varies from 60% (exon 1C) to 10% (exon 1E). This suggests that sequences within the first exons alter the probability that the mRNA will either assemble into polysomes or into translationally inactive mRNP particles.

Animals↗

PubMed1999

Uncoupling protein-3 gene expression: reduced skeletal muscle mRNA in obese humans during pronounced weight loss.

AIMS: Uncoupling protein-3 is a member of a protein family that serves to dissipate energy in the form of heat thereby modulating energy expenditure. Alternative processing of uncoupling protein-3 transcripts results in two mRNA species that encode a large and small protein, perhaps differing in functional activity. Since obesity is associated with disrupted energy homeostasis, we measured muscle mRNA expression in morbidly obese and lean subjects. METHODS: The two uncoupling protein-3 mRNA species were quantified in muscle tissue using an RNase protection assay. Gene locus effects on mRNA expression were studied by quantitative allele-specific primer extension. RESULTS: In both obese and lean subjects, the mRNA species encoding the small protein isoform was twice as abundant as the mRNA species encoding the large protein isoform. Neither the total uncoupling protein-3 mRNA expression nor the molar abundance ratios of the two mRNA species differed between obese and lean male or female subjects. Women who had lost 37+/-22 kg of weight in response to dietary restriction and continued a hypocaloric diet displayed lower mRNA than obese (p<0.005) or lean women (p<0.05). Primer extension assays in lean and obese subjects showed similar allelic mRNA abundance in all but one subject studied. CONCLUSION: Muscle expression of the two uncoupling protein-3 mRNA species is similar in obese and lean people. In obese patients, prolonged hypocaloric diet downregulates uncoupling protein-3 mRNA expression in muscle and can thereby enhance its energy efficiency. Sequence substitutions at the gene locus may only be minor determinants of mRNA expression in muscle tissue.

Adult↗

PubMed1992

Protein kinase C isoforms in human neutrophil cytoplasts.

Granule-poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respiratory burst oxidase, were investigated for their content of several isoforms of protein kinase C (PKC). Immunoblotting with isoform-specific rabbit antibodies (Abs) to PKC revealed that both the alpha-specific and the beta(I and II)- specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The gamma-specific antiserum did not detect any protein of this molecular mass. The cytoplast beta-PKC band was more readily detected than the cytoplast alpha-PKC band. Antibodies to beta I- or beta-II- specific PKC sequences showed the beta II subtype to be the predominant form of beta-PKC, although some beta I was also found. The identity of the 78-kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast alpha- and beta-PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain alpha- and beta-PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast alpha- and beta-PKC. To our knowledge, this is the first demonstration that human neutrophil-derived cytoplasts contain alpha, beta I, and beta II forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In addition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are unable to detect with our alpha, beta, and gamma antibodies. Finally, the granule-poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.

Amino Acid Sequence↗

PubMed1995

Identification and cellular localization of protein kinase C isoforms in cultures of rat type-1 astrocytes.

We have examined which isoforms of protein kinase C were present in rat brain astrocytes and found that: (1) the total of calcium-independent isoforms was greater than the total of calcium-dependent isoforms; (2) there were differences in the intracellular distribution of different isoforms; and (3) the abundance of total protein kinase C was greater in astrocytes from cortex than astrocytes from diencephalon.

Animals↗

PubMed2004

A novel protein derived from the MUC1 gene by alternative splicing and frameshifting.

Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.

Alternative Splicing↗

PubMed1999

Alternative processing of the human and mouse raly genes(1).

A human homolog(RALY) of the mouse Raly gene was isolated and sequenced, and shown to encode a novel protein isoform containing a 16 amino acid in-frame insert in the variable region of the protein. Analysis of the corresponding region of the mouse Raly gene demonstrated that this novel protein isoform is also present in the mouse. Comparative analysis of RALY cDNA and EST sequences suggests the presence of additional alternatively processed RALY transcripts. As in the mouse, the human RALY gene is widely expressed as a 1.7-kb transcript.

Alternative Splicing↗

PubMed2002

The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells.

Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-beta1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H(2)O(2) +/- EGF, PKC, or Ca(2+) modulators. Other cells were transfected to over-express PKC-beta1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H(2)O(2). In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-beta1, enhanced (45)Ca(2+) efflux, normalized Ca(2+), decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-beta1 (+3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-beta1. Antisense inhibition of PKC-beta1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca(2+)-ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-beta1. EGF normalizes Ca(2+) by enhancing Ca(2+) efflux through PKC-beta1. We have identified novel biologic functions, protection of actin and Ca(2+) homeostasis, among the classical isoforms of PKC.

Actins↗

PubMed2005

A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin.

BACKGROUND: Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus. RESULTS: In this report, we identify a novel protein isoform of EB-1/AIDA-1, termed AIDA-1c, that interacts with the CB marker protein, coilin. Northern and nested PCR experiments reveal that the AIDA-1c isoform is expressed in brain and several cancer cell lines. Competition binding experiments demonstrate that AIDA-1c competes with SmB' for coilin binding sites, but does not bind SMN. When ectopically expressed, AIDA-1c is predominantly nuclear with no obvious accumulations in CBs. Interestingly, another EB-1/AIDA-1 nuclear isoform, AIDA-1a, does not bind coilin in vivo as efficiently as AIDA-1c. Knockdown of EB-1/AIDA-1 isoforms by siRNA altered Cajal body organization and reduced cell viability. CONCLUSION: These data suggest that specific EB-1/AIDA-1 isoforms, such as AIDA-1c, may participate in the regulation of nucleoplasmic coilin protein interactions in neuronal and transformed cells.

Binding, Competitive↗

PubMed1999

Differential cellular and subcellular localization of protein phosphatase 1 isoforms in brain.

Protein phosphatase 1 (PP1) is a gene family with a number of important functions in brain. Association with a wide variety of regulatory/targeting subunits is thought to be instrumental in directing the phosphatase to specific subcellular locations and substrates. By using antibodies directed against specific PP1 isoforms, we asked whether PP1 isoforms are differentially distributed in brain. Immunoblotting detects in brain the PP1gamma2 isoform, which had previously been thought to be testis specific, in addition to alpha, beta, and gamma1 isoforms. PP1 isoform expression varies modestly in extracts from different subdissected brain regions and is relatively constant during postnatal development, except for an about twofold increase in PP1gamma2. By immunohistochemical analyses of rat brain, PP1beta and PP1gamma1 cellular expression is widespread but quite distinct from one another. Subcellular fractionation studies demonstrate that PP1beta and PP1gamma1 are selectively associated with different cytoskeletal elements: PP1beta with microtubules, PP1gamma1 with the actin cytoskeleton. Double-immunofluorescence labeling of cultured cortical neurons further reveals a strikingly different and nonoverlapping localization of PP1beta and PP1gamma1: whereas PP1beta localizes to a discrete area of the soma, PP1gamma1 is highly enriched in dendritic spines and presynaptic terminals of cultured neurons. These results show that PP1 isoforms are targeted to different neuronal cytoskeletal compartments with a high degree of specificity, presumably by isoform-specific association with regulatory/targeting proteins. Furthermore, the synaptic localization of PP1gamma1 indicates that it is this isoform that is involved in the regulation of synaptic phosphoproteins such as neurotransmitter receptors and ion channels implicated in synaptic plasticity.

Animals↗