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)

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.
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PMID:Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists. 301 42

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.
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PMID:Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3. 302 30

Stimulation of serine protein kinase activity (referred to as S6 kinase) occurs within minutes of addition of nerve growth factor (NGF) to PC12 rat pheochromocytoma cells. This enzyme activity is not related to the cAMP-dependent protein kinase (protein kinase A) or the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), two other protein kinases potentially involved in signal transduction. Two peaks of NGF-stimulated S6 phosphotransferase activity are observed upon ion exchange chromatography; one that comigrates with the serine kinase previously described in chicken embryo fibroblasts and another with distinct elution properties. Several other factors are also found to regulate S6 phosphotransferase activity in PC12 cells including epidermal growth factor, insulin, and phorbol myristate acetate. Dibutyryl cAMP stimulates S6 phosphotransferase activity; however, this activity is strongly inhibited by the protein kinase A heat stable inhibitor. At least two mechanisms exist through which the NGF-stimulated S6 kinase activity can be regulated, one that apparently can use protein kinase C whereas the other(s) does not. The potential roles of these protein kinase activities in signal transduction and regulation of cell growth and differentiation is discussed.
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PMID:Regulation of protein kinase activities in PC12 pheochromocytoma cells. 303 Jul 27

The treatment of PC12 cells with either nerve growth factor or phorbol 12-myristate 13-acetate caused a decrease in the phosphorylation of a soluble 100-kDa protein (Nsp100). After treatment with nerve growth factor, the activity of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the cytosol was increased. When the cytosol from untreated PC12 cells was preincubated with purified protein kinase C and its cofactors, the phosphorylation of Nsp100 was decreased. The preincubation of cytosol from nerve growth factor-treated PC12 cells with protein kinase C did not decrease Nsp100 phosphorylation further. Moreover, preincubation of partially purified Nsp100 kinase with protein kinase C decreased its ability to phosphorylate Nsp100. These results suggest that the binding of nerve growth factor to its receptor on PC12 cells causes an increase in the activity of protein kinase C in the cytosol and phosphorylation of Nsp100 kinase, which in turn lowers its ability to phosphorylate Nsp100.
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PMID:Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells. 345

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.
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PMID:Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. 358 24

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.
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PMID:A nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells. 377 74

In previous studies from this laboratory (Yu, M.W., Tolson, N. W., and Guroff, G. (1980) J. Biol. Chem. 255, 10481-10492) nerve growth factor treatment of PC12 cells was shown to increase the phosphorylation of a specific nonhistone nuclear protein. In the present work these whole-cell observations have been pursued and a cell-free system developed, based on the detergent treatment devised by Lenk et al. (Lenk, R., Ransom, L., Kaufmann, Y., and Penman, S. (1977) Cell 10, 67-78), in order to explore the nerve growth factor-sensitive phosphorylation system in biochemical detail. Using this preparation it has been shown that treatment of the whole cells with nerve growth factor for 30 min or more leads to a marked increase in the subsequent cell-free phosphorylation of the same nonhistone nuclear protein. A characterization of this phosphorylation indicates that it is quite labile to heat and to structural disruption, that it prefers ATP as phosphate donor, and that it requires Mg2+, but is inhibited by high Mg2+ levels as well as by certain other divalent cations. The site of phosphorylation appears to be on serine residues of the protein, as was the phosphorylation observed previously in whole cells. The use of various inhibitors and stimulators suggests that the kinase catalyzing this phosphorylation is not cAMP-dependent, nor is it similar to protein kinase C or casein kinase. The increased phosphorylation produced by nerve growth factor is not transient, the stimulation being constant for at least 3 days in the continuous presence of nerve growth factor. Increases in the phosphorylation of the same nuclear protein can be seen upon treatment of the cells with other effectors such as epidermal growth factor and dibutyryl cyclic AMP, the latter in spite of the fact that cAMP-dependence could not be established in the cell-free system. Finally, a similar system, with a similar stimulation of phosphorylation due to nerve growth factor treatment, can be prepared from sympathetic ganglia from neonatal animals.
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PMID:Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells. 399 94

In the presence of nerve growth factor (NGF) PC12 pheochromocytoma cells develop properties of sympathetic neurons and extend long microtubule-containing neurites. PC12 cell microtubule protein was isolated using an assembly and disassembly procedure, either directly from an unlabeled cell extract or by copolymerization of a [35S]-methionine-labeled cell extract with rat brain microtubule proteins. Microtubule proteins of PC12 cells treated and untreated with NGF did not reveal any differences as analyzed by SDS-PAGE. The major constituents were the tubulin dimer and microtubule-associated proteins, mainly one high molecular weight component (HMW2) (Mr = 290,000), which resembles by several criteria the high molecular weight protein MAP2 of rat brain microtubule protein. PC12 cell microtubule protein contained intrinsic cAMP-dependent and cAMP-independent protein kinase activity. In vitro phosphorylation experiments indicated that, unlike in rat brain microtubule preparations, both subunits of the tubulin dimer were substrate for the endogenous kinase activity. The addition of cAMP to the incubation medium exerted a slight increase in phosphorylation of the HMW2 protein and an 80,000 polypeptide, and most effectively the phosphorylation of a Mr = 62,000 peptide was increased. This component could be identified as tyrosine hydroxylase by indirect immunoprecipitation procedures. Thus, the in vitro phosphorylation pattern of PC12 cell microtubule protein differed substantially from the one obtained from rat brain microtubule preparations.
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PMID:Microtubule proteins in PC12 pheochromocytoma cells. Isolation, composition and in vitro phosphorylation. 632 Nov 30

Colchicine, nocodazol, and vinblastine, three microtubule-disrupting drugs, were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell-secreted NGF protein in L929 cells, with levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubule-stabilizing agent Taxotere, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down-regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene.
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PMID:Expression of the nerve growth factor gene is controlled by the microtubule network. 747 77

Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.
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PMID:Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. 748 20


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