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PubMed1997

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

Base Sequence↗

PubMed2005

LOCSVMPSI: a web server for subcellular localization of eukaryotic proteins using SVM and profile of PSI-BLAST.

Subcellular location of a protein is one of the key functional characters as proteins must be localized correctly at the subcellular level to have normal biological function. In this paper, a novel method named LOCSVMPSI has been introduced, which is based on the support vector machine (SVM) and the position-specific scoring matrix generated from profiles of PSI-BLAST. With a jackknife test on the RH2427 data set, LOCSVMPSI achieved a high overall prediction accuracy of 90.2%, which is higher than the prediction results by SubLoc and ESLpred on this data set. In addition, prediction performance of LOCSVMPSI was evaluated with 5-fold cross validation test on the PK7579 data set and the prediction results were consistently better than the previous method based on several SVMs using composition of both amino acids and amino acid pairs. Further test on the SWISSPROT new-unique data set showed that LOCSVMPSI also performed better than some widely used prediction methods, such as PSORTII, TargetP and LOCnet. All these results indicate that LOCSVMPSI is a powerful tool for the prediction of eukaryotic protein subcellular localization. An online web server (current version is 1.3) based on this method has been developed and is freely available to both academic and commercial users, which can be accessed by at http://Bioinformatics.ustc.edu.cn/LOCSVMPSI/LOCSVMPSI.php.

Artificial Intelligence↗

PubMed1983

Subcellular localization in normal and vitamin A-deficient rat liver of vitamin A serum transport proteins, albumin, ceruloplasmin and class I major histocompatibility antigens.

The subcellular localization in rat liver cells of retinol-binding protein (RBP), prealbumin, ceruloplasmin, albumin, and class I transplantation antigen chains was investigated by radioimmunoassay determinations. The concentration of RBP was high in the rough and smooth endoplasmic reticulum (SER). The relative concentrations of prealbumin, ceruloplasmin and albumin were similar in the endoplasmic reticulum fractions and in the Golgi fraction. Neither of the proteins were found in significant amounts in the post-microsomal supernatant nor in the plasma membrane. The concentrations of the class I transplantation antigen chains were higher in the Golgi fraction than in the endoplasmic reticulum fractions. In the rough endoplasmic reticulum (RER) fraction ceruloplasmin and the class I antigens partially interact with high-molecular weight (MW) components, presumably membrane-bound glycosyltransferases. RBP, prealbumin and albumin seemed to be present in free form within the microsomal lumen. In vitamin A deficiency the RBP and to a lesser extent the prealbumin concentrations in the endoplasmic reticulum fractions were significantly increased, as compared to fractions from normal livers. This suggests that the presence of vitamin A is a prerequisite for the transport of RBP from the endoplasmic reticulum to the Golgi complex. The intracellular concentrations of albumin and ceruloplasmin were not significantly altered by vitamin A deficiency. In contrast, the amounts of the class I antigen heavy chains were found to be increased.

Animals↗

PubMed1984

Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein.

Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein. 80% of this flavoprotein, as well as all the b-cytochrome, was absent in these fractions from 2 CGD patients, although these patients had normal quantities of FAD in the fractions containing plasma membranes and cytosol. During stimulation the b-cytochrome-associated flavoprotein of the granules translocates with the b-cytochrome to the plasma membrane where NADPH oxidase is localized. Definition of the role of these NADPH oxidase constituents may provide a molecular description of the normal neutrophil respiratory burst and the molecular defect(s) in CGD.

Cell Fractionation↗

PubMed1995

Subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis in the batrachoidid fishes Opsanus beta, Opsanus tau and Porichthys notatus

The subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis were examined in three representatives of fishes of the family Batrachoididae, the gulf toadfish (Opsanus beta), the oyster toadfish (Opsanus tau) and the plainfin midshipman (Porichthys notatus). The primary objective of the study was to compare the biochemical characteristics of these fishes, which represent a range between ammoniotelism and ureotelism (O. beta being facultatively ureotelic), with previous patterns observed for an ammoniotelic teleost (Micropterus salmoides, the largemouth bass) and an obligate ureogenic elasmobranch (Squalus acanthias, the dogfish shark). The present study documents the expression of mitochondrial carbamoyl phosphate synthetase (CPSase) III and cytosolic CPSase II (and its associated enzymes of pyrimidine synthesis, dihydro-orotase and aspartate carbamoyltransferase) in the livers of all three batrachoidid species. Both mitochondrial and cytosolic activities of arginase were present in the livers of all three species, as were cytosolic glutamine synthetase and argininosuccinate synthetase and lyase. However, O. beta also showed mitochondrial glutamine synthetase activity and higher total hepatic levels of glutamine synthetase than either O. tau or P. notatus. Taken together, these observations confirm that the arrangement of these enzymes in the batrachoidid fishes has greater similarity to that of M. salmoides than to that of S. acanthias. However, differences within the family appear to coincide with the different nitrogen excretion strategies. O. tau and P. notatus are primarily ammoniotelic and most closely resemble the ammoniotelic M. salmoides, whereas ureotelism in O. beta is correlated with the presence of a mitochondrial glutamine synthetase and the ability to induce higher total glutamine synthetase activities than O. tau or P. notatus. Additionally, isolated mitochondria from O. beta were able to generate citrulline from glutamine, whereas those from O. tau were not. Also in contrast to S. acanthias, glutamine synthetase activities in the mitochondria of O. beta are consistently lower than those of CPSase III. This and other kinetic observations lend support to the hypothesis that glutamine synthetase may be an important regulatory control point in determining rates of ureogenesis in O. beta.

Journal Article↗

PubMed2004

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Tissue distribution and subcellular localization of a G-protein activated phosphoinositide 3-kinase. An immunohistochemical study.

We studied by light-and electron microscopic immunohistochemical techniques the anatomical distribution and subcellular localization of a G-protein activated phosphoinositide 3-kinase gamma (PI3Kgamma) in several tissues of rat, mouse, dog and man. Using monospecific polyclonal antisera and monoclonal antibodies to the enzyme protein PI3Kgamma immunoreactivity was detected in rat and mouse neutrophils, prostate, kidney, exocrine pancreas, salivary glands (parotid gland) as well as in dog and human exocrine pancreas and parotid gland. No immunoreactive material was found in Langerhans islet cells and in the nervous tissue. Fine structural analysis of PI3Kgamma immunoreactivity revealed immunolabeling of the basolateral membrane of rat kidney tubular cells and certain cells in the parenchyma of the glandular part of rat prostate gland. Our morphological data suggest a possible involvement of the enzyme in cellular transport phenomena and a regulatory influence in secretory processes.

Animals↗

PubMed1975

Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development.

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.

Acetate-CoA Ligase↗

PubMed1970

Subcellular Localization of Salmonella enteritidis Endotoxin in Liver and Spleen of Mice and Rats.

Salmonella enteritidis(14)C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.

Journal Article↗

PubMed2000

Windbeutel is required for function and correct subcellular localization of the Drosophila patterning protein Pipe.

Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered subcellular localization in the follicle cells of females mutant for windbeutel. Thus, Windbeutel protein enables the correct subcellular distribution of Pipe to facilitate its pattern-forming activity.

Animals↗

PubMed2005

The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neurones.

A recent report on the mechanism of synaptic targeting of Ca(v)2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Ca(v)2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Ca(v)2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Ca(v)2.1, and HS and HL encode Ca(v)2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Ca(v)2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Ca(v)2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Ca(v)2.1 channel is different from that of Ca(v)2.2, though these channels share various structural and functional characteristics.

Amino Acid Sequence↗

PubMed2000

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Animals↗

PubMed2003

Subcellular localization of soybean 7S globulin in HepG2 cells and LDL receptor up-regulation by its alpha' constituent subunit.

The aims of this work were to monitor the subcellular localization of soybean 7S globulin in HepG2 cells and determine its interaction with cell protein components, by using laser-induced fluorescence capillary electrophoresis (LIF-CE). Furthermore, we evaluated in the same cell line the involvement of the alpha' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the alpha' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the alpha' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of alpha' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.

Antigens, Plant↗

PubMed2003

A new hybrid approach to predict subcellular localization of proteins by incorporating gene ontology.

Based on the recent development in the gene ontology and functional domain databases, a new hybridization approach is developed for predicting protein subcellular location by combining the gene product, functional domain, and quasi-sequence-order effects. As a showcase, the same prokaryotic and eukaryotic datasets, which were studied by many previous investigators, are used for demonstration. The overall success rate by the jackknife test for the prokaryotic set is 94.7% and that for the eukaryotic set 92.9%. These are so far the highest success rates achieved for the two datasets by following a rigorous cross-validation test procedure, suggesting that such a hybrid approach may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology. The very high success rates also reflect the fact that the subcellular localization of a protein is closely correlated with: (1). the biological objective to which the gene or gene product contributes, (2). the biochemical activity of a gene product, and (3). the place in the cell where a gene product is active.

Algorithms↗

PubMed1994

Sendai virus infection changes the subcellular localization of tryptase Clara in rat bronchiolar epithelial cells.

Tryptase Clara activates the infectivity of Sendai and influenza viruses proteolytically. In this study, we investigated changes in the subcellular localization of tryptase Clara in rat bronchioles with progression of Sendai virus infection. Tryptase Clara and Sendai virus F2 antigen were localized by light and electron immunohistochemical studies. In the uninfected rat lung, tryptase Clara was specifically localized in the secretory granules of respiratory bronchiolar epithelial nonciliated cells, but not in bronchiolar ciliated, or alveolar cells. In the initial stage of Sendai virus infection with slight pathological changes, however, anti-tryptase Clara was highly reactive in luminal peripheral membranes of both nonciliated and ciliated epithelial cells of the bronchioles together with some Sendai virus envelope glycoprotein, F2 antigen. In the progressed stage, tryptase Clara was hard to detect, with heavy accumulation of F2 antigen in the epithelial cells. These immunohistochemical results support our previous findings that in the bronchial lavage fluid tryptase Clara is significantly increased both in amount and activity after viral infection. These results suggest that Sendai virus stimulates the secretion of tryptase Clara from nonciliated bronchiolar epithelial cells to the airway lumen. Accumulation of tryptase Clara on the luminal surface of the bronchiolar epithelial cells and/or in the airway lumen may produce favourable conditions for proteolytic viral activation and multiplication.

Animals↗

PubMed1999

Subcellular localization of GFP-myosin-V in live mouse melanocytes.

Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.

Actin Cytoskeleton↗

PubMed1975

Cell proliferation and subcellular localization of alkaline phosphatase activity in rat liver parenchyma during azo dye carcinogenesis.

A combined method of phosphatase histochemistry and (3H)thymidine radioautography was devised to study the subcellular localization of alkaline phosphatase (AP) activiity with changing pattern of cell proliferation in precancerous livers of rats fed dimethylaminoazobenzene. After 50 hr of continuous infusion of (3H)thymidine into the rats, labeled liver tissue were fixed in glutaraldehyde. Sections were incubated for AP activity in a lead citrate medium (pH 9.4) with beta-glycerophosphate as substrate. Light and electron microscopic examinations of radioautographs revealed that focal groups of 3H-labeled hepatocytes within hyperplastic nodules were coincident to hyperbasophilic foci and distinguishable from the surrounding parenchyma, which was sparsely labeled. Proliferative hepatocytes in the foci exhibited enzyme reaction product indicative of AP activity along the entire surface membranes. The surface AP tography was in contrast to that of the surrounding hyperplastic parencyma, in which regenerative hepatocytes showed a normal localization of AP activity at the bile canalicular membranes. The L-phenylalanine-sensitive snd heat-resistant activity of hyperbasophilic hepatocytes was different from that of normal hepatocytes. The surface enzyme differentiation was accompanied by a decrease of cytoplasmic AP. Golgi elements apparently function in the mobilization of AP into the surface membranes. The phenomena of AP alterations might be related to the abnormal control of cell proliferation and cytodifferentiation leading to malignant growth.

Alkaline Phosphatase↗

PubMed2000

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization.

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.

Amino Acid Sequence↗