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PubMed2003

Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes.

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.

3T3 Cells↗

PubMed2001

Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium.

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.

5' Untranslated Regions↗

PubMed1994

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms.

Minimal ectopic expression of the p58GTA protein kinase results in a provocative phenotype involving cell cycle delay, mitotic catastrophe, and decreased cell viability. In addition, this kinase is well conserved evolutionarily, ubiquitously expressed, and its genes map to a position on human chromosome 1 frequently deleted in the late stages of tumorigenesis. Here we report that the p58GTA protein kinase is a member of a larger subfamily of proteins. The mRNAs encoding these proteins are generated by alternative splicing from multiple duplicated genes. These isoforms range in size from 50 to 110 kDa. Divergence between the alternatively spliced isoforms is localized to the amino-terminal region of the molecule. The entire p58GTA open reading frame is conserved in most of these p58GTA isoforms. The predicted sequences of the larger isoforms encode bipartite nuclear localization signal sequences and extensive polyglutamic acid domains. Antibodies to the p58GTA isoform were used to confirm the presence of the alternatively spliced isoforms in different cell types as well as identify two additional isoforms that appear to arise from a separate gene(s). Cellular fractionation studies indicate that one of the isoforms is found only in the nucleus, and the remainder are found in both the cytoplasm and the nucleus. Expression and localization of some p58GTA isoforms suggest that they may have specialized cellular functions. Because of the large number of isoforms generated from multiple genes we propose naming these kinases PITSLRE alpha 1, alpha 2-1, alpha 2-2, alpha 2-3 alpha 2-4, beta 1, beta 2-1, and beta 2-2 based on the conserved sequence of the PSTAIRE box unique to p34cdc2 kinases and the gene from which they are transcribed.

Alternative Splicing↗

PubMed2003

Translational control of SCL-isoform expression in hematopoietic lineage choice.

We investigated the translational regulation of SCL protein expression and its role in hematopoietic lineage choice. We show that the expression of different SCL protein isoforms is regulated by signal transduction pathways that modulate translation initiation factor (eIF) function. A conserved small upstream open reading frame (uORF) in SCL transcripts acts as a cis-regulatory element for isoform expression. At the onset of erythroid differentiation, truncated SCL protein isoforms arise by alternative translation initiation and favor the erythroid lineage. In comparison, full-length SCL proteins are more efficient at enhancing the megakaryocyte lineage. Together, our studies unravel translational control as a novel mechanism regulating hematopoietic outcome.

Animals↗

PubMed1996

Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function.

Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKC epsilon and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKC epsilon within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC-binding protein. The binding of PKC epsilon to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKC epsilon. Once established, this protein-protein interaction securely anchored PKC epsilon to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKC epsilon isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.

Actins↗

PubMed1999

Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.

Mutations in the human tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some mutations, including mutations in intron 10, induce increased levels of the functionally normal four-repeat tau protein isoform, leading to neurodegeneration. We generated transgenic mice that overexpress the four-repeat human tau protein isoform specifically in neurons. The transgenic mice developed axonal degeneration in brain and spinal cord. In the model, axonal dilations with accumulation of neurofilaments, mitochondria, and vesicles were documented. The axonopathy and the accompanying dysfunctional sensorimotor capacities were transgene-dosage related. These findings proved that merely increasing the concentration of the four-repeat tau protein isoform is sufficient to injure neurons in the central nervous system, without formation of intraneuronal neurofibrillary tangles. Evidence for astrogliosis and ubiquitination of accumulated proteins in the dilated part of the axon supported this conclusion. This transgenic model, overexpressing the longest isoform of human tau protein, recapitulates features of known neurodegenerative diseases, including Alzheimer's disease and other tauopathies. The model makes it possible to study the interaction with additional factors, to be incorporated genetically, or with other biological triggers that are implicated in neurodegeneration.

Animals↗

PubMed1997

Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency.

This study tested for the hypothesis that mechanical loading, not stimulation frequency per se, plays a key role in determining the plasticity of myosin heavy chain (MHC) protein isoform expression in muscle undergoing resistance training. Female Sprague-Dawley rats were randomly assigned to resistance-training programs that employed active 1) shortening (n = 7) or 2) lengthening contractions (n = 8). The medial gastrocnemius (MG) muscles in each group trained under loading conditions that approximated 90-95% of maximum isometric tetanic tension but were stimulated at frequencies of 100 and approximately 25 Hz, respectively. Lengthening and shortening contractions were produced by using a Cambridge ergometer system. The MG muscles trained every other day, performing a total of 16 training session. Both training programs produced significant (P < 0.01) and similar reductions in the fast type IIB MHC protein isoform in the white MG muscle, reducing its relative content to approximately 50% of the total MHC protein isoform pool. These changes were accompanied by increases in the relative content of the fast type IIX MHC protein isoform that were of similar magnitude for both groups. The results of this study clearly demonstrate that stimulation frequency does not play a key role in modulating MHC isoform alterations that result from high-resistance training.

Animals↗

PubMed1998

Training effects on the contractile apparatus.

Skeletal muscle is an extremely heterogeneous tissue, composed of a large variety of fibre types. Its dynamical nature is reflected by the ability to adapt to altered functional demands by qualitative alterations in fibre type composition. The molecular basis of this versatility is that specific myofibrillar and Ca2+-regulatory protein isoforms are assembled to functionally specialized fibre types. Based on this diversity, adult muscle fibres are capable of changing their molecular composition by altered gene expression. Myosin heavy chain (MHC) isoforms and their unique expression in 'pure' fibres, as well as their coexpression in 'hybrid' 'fibres' represent the best markers of muscle fibre diversity and adaptive changes. Chronic low-frequency stimulation (CLFS) and endurance training represent highly suitable models for studying the effects of increased neuromuscular activity on myofibrillar protein isoform expression and fibre type composition. Generally, both models induce fast-to-slow transitions in myofibrillar protein isoforms and fibre types. However, the responses to endurance training are quantitatively less pronounced than those in muscles exposed to CLFS. Parallel changes in isoforms of specific myofibrillar or Ca2+-regulatory proteins during the induced fast-to-slow transitions point to the existence of fibre type-specific patterns of gene expression. The fast-to-slow transitions do not proceed in abrupt jumps from one extreme to the other, but occur in a gradual and orderly sequential manner. Depending on the basal protein isoform profile, and hence the position within the fast-slow spectrum, the adaptive ranges of different fibre types vary. However, adaptive ranges not only depend on a particular fibre type, but also are influenced by species-specific properties.

Animals↗

PubMed2005

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.

Amino Acid Sequence↗

PubMed1998

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Alternative Splicing↗

PubMed1998

Cell- and lamina-specific expression and activity-dependent regulation of type II calcium/calmodulin-dependent protein kinase isoforms in monkey visual cortex.

In situ hybridization histochemistry and immunocytochemistry were used to study localization and activity-dependent regulation of alpha, beta, gamma, and delta isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII) and their mRNAs in areas 17 and 18 of normal and monocularly deprived adult macaques. CaMKII-alpha is expressed overall at levels three to four times higher than that of CaMKII-beta and at least 15 times higher than that of CaMKII-gamma and -delta. All isoforms are expressed primarily in pyramidal cells of both areas, especially those of layers II-III, IVA (in area 17), and VI, but are also expressed in nonpyramidal, non-GABAergic cells of layer IV of both areas and in interstitial neurons of the white matter. CaMKII-alpha and -beta are colocalized, suggesting the formation of heteromers. There was no evidence of expression in neuroglial cells. Each isoform has a unique pattern of laminar and sublaminar distribution, but cortical layers or sublayers enriched for one isoform do not correlate with layers receiving inputs only from isoform-specific layers of the lateral geniculate nucleus. CaMKII-alpha and -beta mRNA and protein levels in layer IVC of area 17 are subject to activity-dependent regulation, with brief periods of monocular deprivation caused by intraocular injections of tetrodotoxin leading to a 30% increase in CaMKII-alpha mRNA and a comparable decrease in CaMKII-beta mRNA in deprived ocular dominance columns, especially of layer IVCbeta. Expression in other layers and expression of CaMKII-gamma and delta were unaffected. Changes occurring in layer IVC may influence the formation of heteromers and protect supragranular layers from CaMKII-dependent plasticity in the adult.

Animals↗

PubMed2006

Differential effects of heterochromatin protein 1 isoforms on mitotic chromosome distribution and growth in Dictyostelium discoideum.

Heterochromatin protein 1 (HP1) is a well-characterized heterochromatin component conserved from fission yeast to humans. We identified three HP1-like genes (hcpA, hcpB, and hcpC) in the Dictyostelium discoideum genome. Two of these (hcpA and hcpB) are expressed, and the proteins colocalized as green fluorescent protein (GFP) fusion proteins in one major cluster at the nuclear periphery that was also characterized by histone H3 lysine 9 dimethylation, a histone modification so far not described for Dictyostelium. The data strongly suggest that this cluster represents the centromeres. Both single-knockout strains displayed only subtle phenotypes, suggesting that both isoforms have largely overlapping functions. In contrast, disruption of both isoforms appeared to be lethal. Furthermore, overexpression of a C-terminally truncated form of HcpA resulted in phenotypically distinct growth defects that were characterized by a strong decrease in cell viability. Although genetic evidence implies functional redundancy, overexpression of GFP-HcpA, but not GFP-HcpB, caused growth defects that were accompanied by an increase in the frequency of atypic anaphase bridges. Our data indicate that Dictyostelium discoideum cells are sensitive to changes in HcpA and HcpB protein levels and that the two isoforms display different in vivo and in vitro affinities for each other. Since the RNA interference (RNAi) machinery is frequently involved in chromatin remodeling, we analyzed if knockouts of RNAi components influenced the localization of H3K9 dimethylation and HP1 isoforms in Dictyostelium. Interestingly, heterochromatin organization appeared to be independent of functional RNAi.

Amino Acid Motifs↗

PubMed1995

FGF-8 isoforms differ in NIH3T3 cell transforming potential.

We previously identified Fgf-8 as a frequently activated gene in tumors from mouse mammary tumor virus-infected Wnt-1 transgenic mice, suggesting that Fgf-8 is a proto-oncogene. We further determined that multiple, secreted protein isoforms that differ at their mature amino termini are encoded by alternatively spliced mRNAs transcribed from the gene. We now present evidence that there are differences in the potency of NIH3T3 cell transformation displayed by three of the FGF (fibroblast growth factor)-8 isoforms. We find that stable transfection of a cDNA for the FGF-8b isoform leads to marked morphological transformation of NIH3T3 cells and rapid tumorigenicity of the transfected cells in nude mice. In contrast, transfection of a cDNA for the FGF-8a or FGF-8c isoform results in moderate morphological changes in the NIH3T3 cells, and the transfected cells are weakly tumorigenic in nude mice. All three transfections result in cells that express comparable amounts of Fgf-8 mRNA and that produce the FGF-8 protein isoforms. The morphological changes observed in NIH3T3 cells can be reproduced by the addition of recombinant FGF-8 protein isoforms to the culture medium. Therefore, these results indicate that there are differences in the potency of transformation of NIH3T3 cells by FGF-8 protein isoforms and suggest that these FGF-8 isoforms may have different in vivo functions.

3T3 Cells↗

PubMed2004

Cell type-specific occurrence of caveolin-1alpha and -1beta in the lung caused by expression of distinct mRNAs.

Two isoforms of caveolin-1, alpha and beta, had been thought to be generated by alternative translation initiation of an mRNA (FL mRNA), but we showed previously that a variant mRNA (5'V mRNA) encodes the beta isoform specifically. In the present study, we demonstrated strong correlation between the expression of the caveolin-1 protein isoforms and mRNA variants in culture cells and the developing mouse lung. The alpha isoform protein and FL mRNA were expressed constantly during the lung development, whereas expression of the beta isoform protein and 5'V mRNA was negligible in the fetal lung before 17.5 days post coitum, and markedly increased simultaneously at 18.5 days post coitum, when the alveolar type I cells started to differentiate. Immunohistochemical analysis revealed the cell type-specific expression of the two isoforms; the alveolar type I cell expresses the beta isoform predominantly, while the endothelium harbors the alpha isoform chiefly. The mutually exclusive expression of caveolin-1 isoforms was verified by Western blotting of the selective plasma membrane preparation obtained from the endothelial and alveolar epithelial cells. The present result indicates that the two caveolin-1 isoforms are generated from distinct mRNAs in vivo and that their production is regulated independently at the transcriptional level. The result also suggests that the alpha and beta isoforms of caveolin-1 may have unique physiological functions.

Animals↗

PubMed2000

Amyloid precursor protein gene isoforms in Alzheimer's disease and other neurodegenerative disorders.

Differential expression of the amyloid precursor protein gene (APP) may be important in the development of amyloidosis in Alzheimer's disease (AD) and experimentally in the brain's response to injury. Controversial data suggests that APP isoforms containing the Kunitz protease inhibitor isoform (APP KPI+) are over expressed in the brains of patients with AD when compared to the non-Kunitz protease inhibitor containing isoforms (APP KPI-). We have investigated this hypothesis using a quantitative analysis of gene expression on brain tissue collected at post-mortem. In situ hybridization has been used with synthetic oligonucleotide probes labelled with 35S to detect the two principal splice variants of APP: APP 695 (KPI-) and APP 751 (KPI+). A prospective brain bank of frozen brain specimens has been established and includes pathologically proven AD (n=15) and other neurodegenerative disorders as controls (n=18). The controls consist of frontal lobe atrophy (n=4), Huntington's disease (n=5), Parkinson's disease (n=4), motor neuron disease (n=2), multi-infarct dementia (n=1), multisystem atrophy (n=1), and subacute sclerosing panencephalitis (n=1). We have observed no significant differences in the expression of APP 695 KPI- mRNA in frontal lobe: 17.49+/-3.26 optical density (OD) units of mRNA expression in AD vs. 16.13+/-1.76 OD units mRNA in controls (P=0.80, linear regression); or temporal lobe: 14.73+/-2.96 in AD vs. 16.49+/-2.15 in controls (P=0.55). No significant differences have been found in APP 751 KPI+ in frontal lobe: 12.86+/-2.98 in AD vs. 13.70+/-2.88 in controls (P=0.97); and temporal lobe: 13.31+/-4.93 in AD vs. 11.07+/-1.99 in controls (P=0. 65). Analysis of the ratios of APP 751 KPI+ OD units of mRNA to APP 695 KPI- mRNA revealed a trend to an increased ratio which did not reach statistical significance: frontal lobe APP 751 KPI+/APP 695 KPI- 1.92+/-1.04 in AD vs. 0.86+/-0.17 in controls (P=0.54); temporal lobe 2.54+/-1.59 in AD vs. 0.96+/-0.11 controls (P=0.34). Our data has not revealed differential expression of APP mRNA isoforms in AD and supports the hypothesis that post-translational events in APP metabolism are important in amyloidogenesis and the pathogenesis of AD.

Adult↗

PubMed1997

Specificity of different isoforms of protein phosphatase-2A and protein phosphatase-2C studied using site-directed mutagenesis of HMG-CoA reductase.

We have expressed the catalytic domain of Chinese hamster HMG-CoA reductase, and 13 point mutations involving the region around the single phosphorylation site for AMP-activated protein kinase. After phosphorylation, these were used to test the specificity of isoforms of protein phosphatase-2A [bovine PP2A(C) (catalytic subunit) and PP2A1 (ABC heterotrimer)] and protein phosphatase-2C (human alpha; mouse alpha, beta1, beta2, beta3, beta4, beta5). PP2A1 had > 50-fold higher activity for HMG-CoA reductase variants than PP2A(C), but their relative selectivity for different variants was similar. Although the specificities of PP2A and PP2C were distinct, no dramatic differences in selectivity were observed between different PP2C isoforms.

AMP-Activated Protein Kinases↗

PubMed1995

Differential expression of protein phosphatase 1 isoforms in mammalian brain.

Rat cDNAs encoding neuronal isoforms of protein phosphatase 1 (PP1) were isolated and their primary structures elucidated. The derived amino acid sequences allowed us to design synthetic C-terminal peptides that were used to raise antibodies. Isoform-specific anti-peptide antibodies against PP1 alpha and PP1 gamma 1 were used to investigate the tissue distribution of PP1 isoforms by immunoblotting. Both isoforms were ubiquitously expressed in mammalian tissues, with the highest levels being observed in brain. Of all neuronal tissues examined, PP1 alpha and PP1 gamma 1 were found to be most abundantly expressed in the striatum. Lesion experiments with kainic acid indicated that both the alpha and the gamma 1 isoforms of protein phosphatase 1 were relatively enriched in the medium-size spiny neurons of the striatum. "In situ" hybridization to rat brain slices using highly sensitive riboprobes also showed PP1 alpha, PP1 beta, and PP1 gamma 1 to be widely expressed in mammalian brain. However, some interesting differences were observed. For example, PP1 alpha and PP1 gamma 1 were found to be expressed in the striatum, where DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000 Da) is also known to be highly expressed. PP1 beta appeared to be relatively less abundant in the same cells, as judged both by "in situ" hybridization and by the apparent absence of PP1 beta clones from the striatal cDNA libraries used.

Amino Acid Sequence↗

PubMed2004

The 14-3-3 protein epsilon isoform expressed in reactive astrocytes in demyelinating lesions of multiple sclerosis binds to vimentin and glial fibrillary acidic protein in cultured human astrocytes.

The 14-3-3 protein family consists of acidic 30-kd proteins expressed at high levels in neurons of the central nervous system. Seven isoforms form a dimeric complex that acts as a molecular chaperone that interacts with key signaling components. Recent studies indicated that the 14-3-3 protein identified in the cerebrospinal fluid of various neurological diseases including multiple sclerosis (MS) is a marker for extensive brain destruction. However, it remains unknown whether the 14-3-3 protein plays an active role in the pathological process of MS. To investigate the differential expression of seven 14-3-3 isoforms in MS lesions, brain tissues of four progressive cases were immunolabeled with a panel of isoform-specific antibodies. Reactive astrocytes in chronic demyelinating lesions intensely expressed beta, epsilon, zeta, eta, and sigma isoforms, among which the epsilon isoform is a highly specific marker for reactive astrocytes. Furthermore, protein overlay, mass spectrometry, immunoprecipitation, and double-immunolabeling analysis showed that the 14-3-3 protein interacts with both vimentin and glial fibrillary acidic protein in cultured human astrocytes. These results suggest that the 14-3-3 protein plays an organizing role in the intermediate filament network in reactive astrocytes at the site of demyelinating lesions in MS.

14-3-3 Proteins↗