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: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser900. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser900, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser900 was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser900 trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the trans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.
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PMID:Steady-state plasma membrane expression of human cytomegalovirus gB is determined by the phosphorylation state of Ser900. 965 12

Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF)-induced luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity) can only be blocked using a selective EGF receptor inhibitor (AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against astrocytoma cells in culture.
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PMID:A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells. 991 10

In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.
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PMID:HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells. 1041 13

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.
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PMID:Differential effects of cAMP in neurons and astrocytes. Role of B-raf. 1046 25

Although the first English-language report of melanoma in 1820 contained a description of a melanoma-prone family, it was 1983 before formal genetic analysis suggested an autosomal dominant mode of inheritance for both melanoma and the then newly described melanoma precursor, dysplastic nevi (DN). Subsequent genetic studies have assumed this model to be correct, although when viewed in aggregate, the data are inconsistent. The first proposed melanoma gene (CMM1) was mapped to chromosome 1p36. This gene assignment has not been confirmed. A second melanoma gene, designated CMM2, has been mapped to chromosome 9p21. This gene assignment has been confirmed, and the cell cycle regulator CDKN2A has been proposed as the candidate gene. Germline mutations in this gene have been identified in about 20% of melanoma-prone families that have been studied to date. Pancreatic cancer occurs excessively in melanoma families with germline mutations in CDKN2A. Germline mutations in the cyclin-dependent kinase gene CDK4 (chromosome 12q14) have been described in three melanoma families. This finding represents a third melanoma gene but one that accounts for only a tiny fraction of all hereditary melanoma. Recently, a familial melanoma-astrocytoma syndrome has been reported. Large germline deletions of 9p21 occur in these families, with the p19 gene implicated in its pathogenesis. At present, clinical predictive genetic testing for mutations in the CDKN2A gene is available commercially, but its use has been limited by uncertainty as to how test results would affect the management of melanoma-prone family members. Currently, management recommendations include monthly skin self-examination, clinical skin examination once or twice yearly, a low threshold for simple excision of changing pigmented lesions, moderation of sun exposure, and appropriate use of sunscreens. A heritable determinant for total nevus number has been suggested by twin studies. Other data suggest the presence of a major gene responsible for "total nevus density" in melanoma-prone families. Approximately 55% of the mole phenotype in multiplex melanoma families was explained by this proposed gene. An autosomal dominant mode of inheritance has been proposed for DN, and data exist to suggest that DN may be a pleiotropic manifestation of the 1p36 familial melanoma gene. However, there clearly are melanoma-prone families that do not express the dysplastic nevus trait, and some of the families linked to CDKN2A also present with dysplastic nevi. Several studies have shown a surprisingly high prevalence of DN on the skin of family members of probands with DN. In light of the extensive evidence documenting that persons with DN (both sporadic and familial) have an increased prospective risk of melanoma, these family studies suggest that relatives of persons with DN should be examined for both DN and melanoma. Genetic determinants play a major role in the pathogenesis of normal nevi, DN, and melanoma. Identifying the molecular basis of these genetic events promises to enhance melanoma risk-reduction strategies and, ultimately, reduce melanoma-associated mortality.
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PMID:The genetics of hereditary melanoma and nevi. 1998 update. 1063 Jan 72

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.
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PMID:Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells. 1069 11

Cannabinoids exert most of their effects in the central nervous system through the CB(1) cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin and wortmannin blocked the CB(1) receptor-evoked activation of PKB, pointing to the sequential involvement of a G(i)/G(o) protein and phosphoinositide 3'-kinase. The functionality of the cannabinoid-induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB(1) receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB(2) receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB(1) receptor.
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PMID:The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt. 1074 65

We investigated Ca(2+)/calmodulin (CaM)-mediated regulation of the desensitizing process of the histamine H(1) receptor-mediated increase in intracellular Ca(2+) concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine-induced Ca(2+) responses in cells pretreated with histamine for 15 s-30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine-mediated internalization of the histamine H(1) receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca(2+) and, even more strikingly, by the presence of W-7 (a CaM antagonist). FK506 and cyclosporin A, Ca(2+)/CaM-dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN-62, a Ca(2+)/CaM-dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W-5, okadaic acid, and KN-04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high-affinity site for histamine was converted to a lower-affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN-62 and FK506. These results provide the first evidence that Ca(2+)/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H(1) receptors.
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PMID:Ca(2+)/calmodulin-mediated regulation of the desensitizing process in G(q) protein-coupled histamine H(1) receptor-mediated Ca(2+) responses in human U373 MG astrocytoma cells. 1089 54

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.
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PMID:Expression of p57(KIP2) potently blocks the growth of human astrocytomas and induces cell senescence. 1098 Jan 31

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.
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PMID:Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock. 1122 58


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