Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.
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PMID:Characteristics of the F52 protein, a MARCKS homologue. 161 55

Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H-myristoylated proteins of 50 and 40-45 kDa were observed in cell extracts heated to > 80 degrees C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced rechange of the protein on nitrocellulose. Addition of beta-12-O-tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.
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PMID:Differential expression of MARCKS and other calmodulin-binding protein kinase C substrates in cultured neuroblastoma and glioma cells. 796 53

Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (IK) and the M-current (IM). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: IK, a small current through a "T-like" Ca2+ channel, and no M-current. Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22 +/- 7%. In contrast to the stimulation of IK observed with cardiac cells, PKC activation produced only a small inhibition of IK, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of IK in neuroblastoma cells.
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PMID:Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C. 832 Jul 19

An 80-kDa protein labeled with [3H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4 beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100-soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of beta-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, beta-TPA-stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to alpha-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.
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PMID:Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells. 845 32

Treatment of 8-Br-cAMP promotes neurite outgrowth and neuronal differentiation in N1E115 mouse neuroblastoma cells. Prior or simultaneous treatment of PMA blocks 8-Br-cAMP-mediated neurite outgrowth. Phosphorylation of cellular proteins during these treatments was examined in a permeabilized cell system. While PMA promotes phosphorylation of the heat-stable protein kinase C substrates MARCKS and neuromodulin, 8-Br-cAMP hastens the dephosphorylation of a protein of M(r)95k (p95). Extensively purified, N-terminal sequenced, and judged from its phosphorylation properties, p95 was identified as the eukaryotic elongation factor-2 (eEF-2), whose dephosphorylation has been reported to be related to an increase in protein synthesis. It is likely 8-Br-cAMP stimulates dephosphorylation of eEf-2, promotes protein synthesis that eventually leads to neuronal differentiation in N1E115 cells.
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PMID:Identification of a rapidly dephosphorylating 95-kDa protein as elongation factor 2 during 8-Br-cAMP treatment of N1E115 neuroblastoma cells. 852

To investigate the regulation of phorbol ester-stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine-rich protein kinase C substrate (MARCKS) and the alpha-isoform of protein kinase C (PKC-alpha) were overexpressed in a human neuroblastoma (SK-N-MC) cell line that does not increase PtdCho synthesis in response to 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl-3H] choline was stimulated 1.88-2.34-fold in the presence of 100-200 nM TPA. In clones overexpressing PKC-alpha (30-40-fold increased level of protein) or in mock-transfected vector controls, TPA had much less of a stimulatory effect (1.04-1.43 fold) on PtdCho synthesis. TPA caused translocation of PKC-alpha and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK-N-MC cells, MARCKS is required for TPA-stimulated synthesis of PtdCho and PKC-alpha alone is insufficient for supporting enhanced synthesis.
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PMID:Overexpression of MARCKS, but not protein kinase C-alpha, increases phorbol ester-stimulated synthesis of phosphatidylcholine in human SK-N-MC neuroblastoma cells. 862 36

Neuroblastoma and glioma cells differentially express isoforms of protein kinase C (PKC) and myristoylated PKC substrates (e.g. MARCKS). Correlation with metabolism of membrane phospholipids suggests that PKC-alpha and MARCKS may be required to mediate phosphatidylcholine turnover stimulated by phorbol ester (beta-TPA). To evaluate relationships to neural cell differentiation, SK-N-SH human neuroblastoma cells were treated with 20 nM beta-TPA. In beta-TPA-treated cells, growth arrest and differentiation occurred (neurite extension; 40-60% decrease in cell number, total protein and RNA). By day 4, mRNA for PKC-alpha and MARCKS increased and, after an initial decrease, PKC-alpha protein also increased. At day 4, phosphatidylcholine synthesis was 3-5 fold greater than in control cells. In contrast, C6 glioma cells treated with beta-TPA showed no growth arrest, decreased PKC-alpha protein (< 20%) and lower phosphatidylcholine synthesis. Thus, induced differentiation of human neuroblastoma cells involved increased expression of PKC-alpha and MARCKS and synthesis of phosphatidylcholine, consistent with involvement of PKC-alpha and MARCKS in regulation of phosphatidylcholine turnover during neurite growth.
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PMID:Protein kinase C isoforms and growth, differentiation and phosphatidylcholine turnover in human neuroblastoma cells. 890 63

We have recently shown that the anti-Parkinson-propargyl-containing monoamine oxidase B (MAO-B) inhibitor drug, rasagiline [N-propargyl-(1R)-aminoindan], and its cholinesterase inhibitor derivatives TV3326 and TV3279, regulate amyloid precursor protein (APP) processing by a protein kinase C (PKC)-dependent mechanism in SH-SY5Y neuroblastoma and PC12 cells. In the present study, we investigated the effect of rasagiline and its derivatives on the regulation of the PKC-dependent mechanism and APP processing under in vivo conditions. Administration of rasagiline (0.1 mg/kg) to male C57/BL mice for 14 days significantly decreased membrane-bound holoprotein APP levels in the hippocampus. Additionally, we observed that rasagiline up-regulated p-PKC levels and the expression of alpha and epsilon PKC isozymes in the hippocampus, indicating that the mechanism by which rasagiline affects APP processing may be related to PKC-associated signalling. The results also demonstrate that rasagiline treatment significantly elevated the levels of phosphorylated myristoylated alanine-rich C kinase substrate (p-MARCKS), a major substrate for PKC, as well as the levels of receptors for activated C kinase 1 (RACK1). Similar effects on APP and PKC levels were also demonstrated for the two cholinesterase inhibitor derivatives of rasagiline, TV3326 and TV3279. These results indicate that rasagiline and its derivatives regulate PKC-dependent mechanisms and APP processing. The activation and induction of PKC and MARCKS by these drugs may have a crucial role not only in their neuroprotective activity, but also in their ability to affect neuronal plasticity and spatial learning processes.
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PMID:Regulation of protein kinase C by the anti-Parkinson drug, MAO-B inhibitor, rasagiline and its derivatives, in vivo. 1514 4

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.
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PMID:Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase zeta. 1605 37

Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.
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PMID:Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice. 2465 76


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