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)

K252a, an efficient serine/threonine protein kinase inhibitor (IC50s of 10 to 30 nM), has been shown to block the neuronal differentiation of rat pheochromocytoma PC12 cells induced by nerve growth factor (NGF). In this report, we demonstrate that K252a is a potent inhibitor (IC50 of 3 nM) of the tyrosine protein kinase activity of the NGF receptor gp140trk, the product of the trk protooncogene. K252a also inhibits the kinase activity of its transforming alleles, the trk oncogenes, and of the related neurotrophin receptors gp145trkB and gp145trkC, the products of the other known members of the trk gene family, trkB and trkC. In contrast, K252a has no effect (even at micromolar concentrations) on other tyrosine protein kinases such as the receptors for EGF and PDGF and the products of the v-src and v-fms oncogenes. In addition, K252a rapidly reverts the transformed phenotype of NIH3T3 cells transformed by either autocrine stimulation of the trk family of receptors by their cognate ligands or by expression of trk oncogenes isolated from human tumors. The selectivity of K252a for the catalytic activity of the trk family of kinases should help to establish the structural basis for the rational design of highly specific tyrosine protein kinase inhibitors.
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PMID:K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. 131 98

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.
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PMID:Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase. 131 57

The protein kinase C (PKC) inhibitor staurosporine, a member of the K252a family of fungal alkaloids that are known as protein kinase inhibitors, induces neurite outgrowth in pheochromocytoma PC12 cells. The progressive staurosporine-induced neurotropic effect (EC50 = 50 nM) has the following characteristics: it is evident after 4 hr of incubation, requires the continuous presence of staurosporine, occurs at 37 degrees but not at 4 degrees, and is not blocked by K252a derivatives. Scanning electron micrographs showed long neurites, ruffling, and dense networks in nerve growth factor (NGF)-treated cells and short neurites, flattening, and smooth cell surface in staurosporine-treated cells. [3H]Staurosporine binding, which was time, temperature, and dose dependent, saturated at 5-10 nM. Other kinase inhibitors were poor competitors. The [3H]staurosporine bound over 20 hr at 37 degrees was poorly dissociated by acetic acid wash or unlabeled staurosporine. These results suggest an uptake process occurring at 37 degrees that is required for the neurotropic effect of staurosporine. NGF did not interfere with staurosporine binding, and staurosporine did not affect NGF receptor binding. At neurotropic concentrations of staurosporine, PKC in PC12 cells was completely inhibited. When PKC activity was down-regulated by prolonged exposure to phorbol myristate acetate, PC12 cells responded to staurosporine with neurite outgrowth similar to that of untreated cells. Although the target and mechanism of the neurotropic effects of staurosporine remain to be determined, the observed effects on PKC-deficient cells indicate that PKC may not be required for the neurotropic effect of this compound in PC12 cells. These results suggest that caution should be taken in the interpretation of staurosporine action in vivo, and they provide a pharmacological tool for the development of potential neurotropic drugs.
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PMID:Staurosporine-induced neurite outgrowth in PC12 cells is independent of protein kinase C inhibition. 163 52

To explore the molecular mechanisms of nerve growth factor (NGF) action, we have attempted to identify proteins that immunoprecipitate with the NGF receptor. An anti-NGF receptor antibody was developed that immunoprecipitated the 75-Kd receptor in PC-12 cells. In [35S]methionine-labeled cells lysed with nonionic detergent, immunoprecipitation with this antireceptor antisera specifically brought down several associated proteins, although prior treatment of cells with NGF produced no apparent change in the distribution of these proteins. However, in vitro phosphorylation assays of the immunoprecipitated complex revealed the presence of a serine kinase that phosphorylated two predominant substrates with Mrs of 60 and 130 Kd. Prior treatment of cells produced no change in the appearance of the 60-Kd phosphoprotein, but NGF did stimulate the appearance of the 130-Kd protein. This effect was observed with as little as 0.1 nM NGF and was maximal at 5 min, but declined thereafter. Prior treatment of cells with NGF did not increase the phosphorylation of enolase added exogenously to the immunoprecipitates, suggesting that this action of NGF may have reflected the hormone-dependent association of the 130-Kd protein with the receptor, rather than activation of a receptor-associated kinase. Thus the association of the NGF 75-Kd receptor with a 130-Kd protein may be involved in signal transduction for the growth factor, although the role of this receptor in the NGF-dependent tyrosine phosphorylation remains unclear.
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PMID:Nerve growth factor induces the association of a 130-Kd phosphoprotein with its receptor in PC-12 pheochromocytoma cells. 166 Mar 8

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

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.
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PMID:Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors. 768 Feb 48

A series of the synthetic protein kinase inhibitors known as tyrphostins were examined for their effects on the tyrosine autophosphorylation of the pp140c-trk, nerve growth factor (NGF) receptor. One of the tyrphostins, AG879, inhibited NGF-dependent pp140c-trk tyrosine phosphorylation, but did not affect tyrosine phosphorylation of epidermal growth factor or platelet-derived growth factor receptors. In addition, the tyrosine phosphorylation of the receptor-associated protein pp38 was also attenuated by the tyrphostin. This effect was time- and dose-dependent, although inhibition of pp38 phosphorylation occurred earlier and at lower concentrations of the compound. AG879 also inhibited NGF-induced PLC-gamma 1 phosphorylation, phosphatidylinositol-3 (PI3) kinase activation, the association of the tyrosine-phosphorylated proteins pp100 and pp110 with the p85 subunit of PI-3 kinase, mitogen activated protein and raf-1 kinases, and c-fos induction. In addition, AG879 inhibited NGF-induced neurite outgrowth in PC12 cells. These data indicate that tyrosine kinase activity of the pp140c-trk NGF receptor is essential for the cellular actions of this growth factor.
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PMID:The tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor. 768 92

Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (+/- NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and approximately 50-80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.
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PMID:A purine analog-sensitive protein kinase activity associates with Trk nerve growth factor receptors. 768 57

The biological activity of nerve growth factor (NGF) has been shown to be mediated by the p140trkA receptor tyrosine kinase, while the role of the p75 NGF receptor (p75NGFR) is still unresolved. Here we have investigated the relative contribution of p140trkA and p75NGFR to early consequences of NGF binding: ligand internalization, p140trkA autophosphorylation, and tyrosine phosphorylation of Shc, phospholipase C gamma-1 (PLC gamma-1), and extracellular signal-regulated kinases (ERKs). It was found that NGF internalization was neither prevented by blocking p140trkA activity using the protein kinase inhibitors methylthioadenosine, staurosporine, and K-252a, nor by inhibiting NGF binding to p75NGFR with antibodies. However, when NGF binding to p140trkA was reduced by the use of a synthetic peptide corresponding to amino acids 36-53 of human p140trkA, internalization of NGF was decreased. Thus, at least in PC12 cells, internalization appears to require binding of NGF to p140trkA, but occurs irrespective of p140trkA kinase activity and ligand occupancy of p75NGFR. The NGF triple mutant Lys-32/Lys-34/Glu-35 to Ala, which has been demonstrated to bind to p140trkA, but not to p75NGFR, induced tyrosine phosphorylation more rapidly than wild-type NGF. Likewise, NGF-induced tyrosine phosphorylation was accelerated when NGF binding to p75NGFR was prevented with REX-IgG. These findings indicate that NGF bindign by p75NGFR may modulate NGF-induced p140trkA kinase activity.
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PMID:p75 nerve growth factor receptor modulates p140trkA kinase activity, but not ligand internalization, in PC12 cells. 781 75

We have used serum-deprived cultures of wild type and genetically modified PC12 cells to investigate the molecular mechanisms by which monosialoganglioside (GM1) rescues neuronal cells from apoptotic death elicited by withdrawal of trophic support. Our findings indicate that GM1-promoted survival can be mediated in part by the Trk NGF receptor as well as by TrkB, and potentially by tyrosine kinase receptors for additional neurotrophic growth factors. Experiments employing K-252a, an inhibitor of Trk kinases, and PC12 cells overexpressing a dominant inhibitory form of Trk both indicate that a portion of the survival-promoting activity of GM1 is evoked by receptor dimerization and autophosphorylation. In consonance with this we find that GM1 stimulates Trk tyrosine autophosphorylation and Trk-associated protein kinase activity. These observations may provide a mechanism to account for the reported in vitro and in vivo trophic actions of GM1.
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PMID:Prevention of apoptotic neuronal death by GM1 ganglioside. Involvement of Trk neurotrophin receptors. 785 88


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