epidermal-growth-factor and staurosporine-aglycone

Tyrosine kinase receptor A (TrkA), the high-affinity receptor of nerve growth factor (NGF), is known to play key roles in ovarian follicular development, such as assembly of early follicles and follicular ovulation. However, little is known about the roles of TrkA in cumulus oocyte complex (COC) expansion. In this study, we found that TrkA was abundant in large antral follicles and knockdown of TrkA in COCs attenuated epidermal growth factor (EGF)-induced COC expansion and further decreased the ovulation rate. The effect of TrkA on COC expansion was not mediated through downstream EGF effectors, phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) or drosophila mothers against decapentaplegic protein (SMAD), or through up-regulation of COC expansion-related transcripts such as prostaglandin-endoperoxide synthase 2 (Ptgs2), hyaluronan synthase 2 (Has2), TNF-induced protein 6 (Tnfaip6) or pentraxin 3 (Ptx3). However, pharmacological blockade of TrkA transducing activity (K252α) in COCs decreased the mRNA expression and protein secretion of interleukin-6 (IL-6), identified from mRNA microarray of K252α-treated COCs. Meanwhile, knockdown of IL-6 attenuated EGF-induced COC expansion. In addition, IL-6 rescued the inhibitory effect of K252α on EGF-induced cumulus expansion. Therefore, IL-6 may act as a new potential cumulus expansion-related transcript, which may be involved in the integration of TrkA and EGF signaling in affecting COC expansion. Here, we provide mechanistic insights into the roles of TrkA in EGF-induced cumulus expansion. Understanding potential cross-points between TrkA and EGF affecting cumulus expansion will help in the discovery of new therapeutic targets in ovulation-related diseases.

Topics: Animals; C-Reactive Protein; Carbazoles; Cell Adhesion Molecules; Cumulus Cells; Cyclooxygenase 2; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Developmental; Glucuronosyltransferase; Hyaluronan Synthases; Indole Alkaloids; Interleukin-6; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Tissue Proteins; Oocytes; Ovulation; Receptor, trkA; RNA, Small Interfering; Signal Transduction; Smad Proteins

We have previously reported that estrogen induced proliferation of urothelial cells is modulated by nerve growth factor (NGF). In this study we investigated whether progesterone induces urothelial cell proliferation and whether this effect is modulated by NGF or by epidermal growth factor (EGF).. Experiments were performed using human urothelial cells immortalized by human papillomavirus E6. Cell proliferation was determined using the alamarBlue (Trek Diagnostic, Westlake, New York) assay. Human papillomavirus were seeded in 48-well plates. They were incubated with 5% alamarBlue and different concentrations of progesterone, EGF or NGF in the presence or absence of neutralizing EGF or NGF antibody, K252a (an inhibitor of trkA, the high affinity receptor for NGF), Ru-486 (an antagonist of progesterone and glucocorticoid receptor) or ZK 137 316 (a specific antagonist of progesterone receptor). Immunoblotting was performed using specific antibodies for progesterone receptor, glucocorticoid receptor or EGF receptor. EGF content in conditioned medium was determined by enzyme-linked immunosorbent assay.. In the presence of 10 nM to 1 microM progesterone urothelial cell proliferation was significantly increased 8.6% to 51.1%. This effect was abolished by ZK137 316 or by Ru-486. Hydrocortisone also induced urothelial cell proliferation. This effect was blocked by Ru-486 but not by ZK137 316. In addition, progesterone stimulated urothelial cell proliferation was inhibited by neutralizing EGF antibody but not by NGF antiserum or K252a. We also found that EGF synthesis and release by urothelial cells was increased by exogenous progesterone. This effect of progesterone was inhibited by ZK 137 316.. These findings indicate that progesterone has the capacity to induce urothelial cell proliferation through its cognate receptor and this effect is mediated by EGF but not by NGF.

Topics: Carbazoles; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Hormone Antagonists; Humans; Hydrocortisone; Indole Alkaloids; Mifepristone; Nerve Growth Factor; Progesterone; Receptors, Estrogen; Steroids; Urothelium

Members of the epidermal growth factor (EGF) family are frequently implicated in the injury response of the mammalian nervous system. Although this implication is supported by extensive molecular evidence, it is not underpinned by conclusive functional data. Recently, we found that expression of an EGF homolog from the pond snail Lymnaea stagnalis (L-EGF) is upregulated after axotomy in the adult CNS, suggesting a role for this molecule in the injury response of the CNS. In the present study we asked whether L-EGF can promote axonal regeneration of three types of identified neurons in organ-cultured CNS. Treatment with purified L-EGF substantially enhanced axonal regeneration of all three types of neurons, an effect inhibited by submicromolar doses of PD153035, a specific EGF receptor (EGFR) tyrosine kinase inhibitor. In addition, PD153035 and K252a, a nonspecific kinase inhibitor, also reduced the degree of axonal regeneration that occurs without L-EGF supplementation, indicating that L-EGF or other EGFR ligands synthesized in the CNS participate in the regenerative response. An intriguing aspect of these results is that axonal regeneration of different, intrinsically L-EGF responsive and unresponsive neurons occurred in a coordinated manner. This observation suggests that indirect in addition to direct actions contribute to the beneficial effect of L-EGF. In conclusion, we provide functional evidence that an EGF homolog can promote axonal regeneration, substantiating existing molecular evidence implicating the EGF family in peripheral nerve regeneration and emphasizes the therapeutic potential of these molecules.

Topics: Animals; Axons; Carbazoles; Central Nervous System; Disease Models, Animal; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Indole Alkaloids; Lymnaea; Nerve Crush; Nerve Regeneration; Neurons; Organ Culture Techniques; Quinazolines; Reproducibility of Results

Differentiation of PC12 cells triggered by nerve growth factor (NGF) is characterized by several well-defined events including induction of a set of neuron-specific genes, gain of membrane excitability, and morphological changes such as neurite outgrowth. Here we report that K252a, a protein kinase inhibitor, converts the proliferation signal of epidermal growth factor (EGF) into the morphological differentiation signal without inducing the sustained activation of ERK and the expression of neurofilament. Major effects of EGF/K252a, found also in the NGF-treated cells, are the sustained mobility shift of paxillin in SDS-PAGE and the promoted association of Crk-II with paxillin. These effects explain the prominent and robust development of peripheral focal adhesion assembly and stress fiber-like structures observed in the early stages of PC12 cell differentiation. These results suggest a model that cytoskeletal reorganization via focal adhesion assembly triggered by NGF provides a signal required for the morphological differentiation of PC12 cells.

Topics: Actin Cytoskeleton; Animals; Carbazoles; Cell Adhesion; Cell Differentiation; Culture Media, Serum-Free; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epidermal Growth Factor; Immunohistochemistry; Indole Alkaloids; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Neurites; Neurons; Paxillin; PC12 Cells; Phosphoproteins; Phosphorylation; Rats; Recombinant Fusion Proteins

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine-1-phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin-like growth factor-1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4-fold increase in Vmax. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.

Topics: Animals; Carbazoles; Carrier Proteins; Cytosol; Enzyme Activation; Epidermal Growth Factor; Growth Substances; Indole Alkaloids; Membrane Proteins; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; PC12 Cells; Pheochromocytoma; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptor, trkA; Time Factors

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

Topics: Alkaloids; Antibodies, Monoclonal; Carbazoles; Carcinogens; Carcinoma; Cell Movement; Dimethyl Sulfoxide; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Indole Alkaloids; Male; Neoplasm Metastasis; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

Epidermal growth factor (EGF) has recently been shown to cause certain strains of PC12 cells to extend short neurites. This EGF-induced differentiation of PC12 was found to be potentiated by the protein kinase inhibitor, K252a, in that PC12 cells treated with both EGF and K252a extended long branched neurites similar to those induced by nerve growth factor (NGF). As reported here no other protein kinase inhibitor or activator mimicked or blocked the effect of K252a on EGF-induced PC12 differentiation. Cyclic adenosine 3',5'-monophosphate (cAMP) also potentiated EGF-induced neurite outgrowth from PC12 cells, but the mechanism of this potentiation was different from that of K252a. Cells that had been exposed to EGF and then stripped of their neurons extended neurites again when retreated with EGF in the absence of RNA synthesis or when treated with NGF in the absence of RNA synthesis. Thus EGF can prime PC12 cells for either EGF or for NGF, a finding that further suggests that EGF and NGF use similar signaling pathways to induced neuronal differentiation of PC12.

Topics: Animals; Bucladesine; Carbazoles; Cell Differentiation; Drug Synergism; Enzyme Activation; Epidermal Growth Factor; Indole Alkaloids; Mitogens; Neurites; PC12 Cells; Phosphoproteins; Protein Kinase Inhibitors; Protein Kinases; Rats; Time Factors

The protein kinase inhibitors K-252a and K-252b have been shown earlier to block the actions of nerve growth factor and other neurotrophins and, at lower concentrations, to selectively potentiate neurotrophin-3 actions. In the present study we show that K-252a, but not K-252b, enhances epidermal growth factor (EGF)-and basic fibroblast growth factor (BFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (Erks) elicited by EGF of bFGF was also increased in the presence of K-252a, and this signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-gamma 1 were not changed. The effect of K-252a on Erks was resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. In partial contrast to the actions of K-252a, the neurotrophin-3-potentiating effect of K-252b was accompanied by an increase in tyrosine phosphorylation of the Erks and of phospholipase C-gamma 1. Finally, although K-252a alone did not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C-gamma 1, this compound alone stimulated phosphatidylinositol hydrolysis. Our findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction of EGF and bFGF. Our results are further compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation.

Topics: Animals; Carbazoles; Cell Differentiation; Choline O-Acetyltransferase; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Immunoblotting; Indole Alkaloids; Nerve Growth Factors; Neurites; PC12 Cells; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein Kinase Inhibitors; Rats; Tetradecanoylphorbol Acetate; Tyrosine

Epidermal growth factor (EGF) induced short neurites in two different strains of PC12 cells. The length of the EGF-induced neurites was markedly increased in the presence of the protein kinase inhibitor K252a, which is known to inhibit differentiation induced by nerve growth factor (NGF). EGF-induced differentiation of PC12 required RNA synthesis and activity of the ras proto-oncogene product. EGF increased the levels of three neurofilament proteins and the mRNA level of two late response genes (SCG10 and 63) known to be induced by NGF. Together, EGF and K252a caused a greater increase in these mRNAs than did either agent alone. K252a did not alter the extent of EGF-induced autophosphorylation of the EGF receptor, but it did decrease the extent of receptor phosphorylation in the absence of added EGF. Thus, the ability of the EGF receptor to trigger neuronal differentiation may depend on the state of its phosphorylation at serine and/or threonine residues. Two other strains of PC12 did not extend neurites when exposed to EGF, even when K252a was also present. Thus, the differentiating effect of EGF on PC12 is PC12 strain-specific.

Topics: Animals; Blotting, Northern; Blotting, Western; Carbazoles; Cell Differentiation; Epidermal Growth Factor; Gene Expression Regulation; Humans; Indole Alkaloids; Mice; Nerve Growth Factors; Neurites; PC12 Cells; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Receptors, Nerve Growth Factor; RNA; Signal Transduction; Tetradecanoylphorbol Acetate

Transforming growth factor alpha messenger RNA and protein levels are highest in the striatum, the target area of mesencephalic dopaminergic neurons of the substantia nigra, suggesting a role as a target-derived neurotrophic factor for these cells. To test this hypothesis, we characterized the actions of transforming growth factor alpha on fetal rat dopaminergic neurons in culture. Transforming growth factor alpha promoted dopamine uptake in a dose- and time-dependent manner. Administration of transforming growth factor alpha at the time of plating for 2 h produced a significant increase in dopamine uptake after five days of growth in vitro. As cultures aged they became less responsive to transforming growth factor alpha, such that longer times of exposure were required to elicit a similar, but weaker, response. Dopaminergic cell survival was selectively promoted by transforming growth factor alpha, since there was an increase in the number of tyrosine hydroxylase-immunostained cells without a parallel increase in the total number of neuron-specific enolase-immunopositive cells. Neurite length, branch number and soma area of tyrosine hydroxylase-immunopositive cells also were enhanced by transforming growth factor alpha treatment. Increases in each of the dopaminergic parameters due to transforming growth factor alpha were accompanied by a rise in glial cell number, making it possible that these effects were mediated by this cell population. The neurotrophin antagonist, K252b, failed to inhibit the transforming growth factor alpha-induced increase in dopamine uptake, indicating that transforming growth factor alpha's effects were not mediated by neurotrophin mechanisms. The actions of transforming growth factor alpha on the differentiation of dopaminergic neurons only partially overlapped with those of epidermal growth factor. Thus, while transforming growth factor alpha and epidermal growth factor are believed to share the same receptor they differentially affect dopaminergic cell development in vitro. These results indicate that transforming growth factor alpha is a trophic factor for mesencephalic cells in culture and suggests that transforming growth factor alpha plays a physiological role in the development of these cells in vivo.

Topics: Animals; Astrocytes; Biomarkers; Carbazoles; Cell Differentiation; Cell Division; Cells, Cultured; Cytarabine; Dopamine; Epidermal Growth Factor; Indole Alkaloids; Insulin-Like Growth Factor I; Mesencephalon; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; Rats; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor alpha; Tyrosine 3-Monooxygenase

Although epidermal growth factor (EGF) and nerve growth factor (NGF) have markedly different biological effects on PC-12 cells, many of the signaling events following ligand binding are similar. Both EGF and NGF result in the induction of the primary response gene egr-1/TIS8 and increased methylation of a variety of membrane-associated proteins as early as 5 min after EGF or NGF treatment using a methylation assay that detects methyl esters as well as methylated arginine residues. At 20 min after stimulation with these factors, the stimulation of methylation by NGF is greater than that of EGF, especially in the polypeptides of 36-42 and 20-22 kDa. To help dissect the pathways involved in these cellular responses, the protein kinase inhibitor K252a and the methyltransferase inhibitor 5'-methylthioadenosine (MTA) were used. Both K252a and MTA inhibit NGF-, but not EGF-mediated, primary response gene expression. In contrast, MTA, but not K252a, can block NGF-induced membrane associated protein methylation. These data suggest a role for differential protein methylation reactions in EGF and NGF signal transduction.

Topics: Animals; Carbazoles; Deoxyadenosines; DNA-Binding Proteins; Early Growth Response Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Immediate-Early Proteins; Indole Alkaloids; Membrane Proteins; Methylation; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; PC12 Cells; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Thionucleosides; Transcription Factors

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Chromatography; Epidermal Growth Factor; Fibroblast Growth Factor 2; Indole Alkaloids; Nerve Growth Factors; Neurites; PC12 Cells; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases

Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP-induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and microfilaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by microfilaments.

Topics: Actins; Animals; Bucladesine; Carbazoles; Colchicine; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; Fibroblast Growth Factor 2; Indole Alkaloids; Isoquinolines; Nerve Growth Factors; Neurons; Protein Kinases; Rats; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The influences of protein kinase inhibitors, K-252a and staurosporine, on the neurotrophic effects of epidermal growth factor (EGF) were investigated in dissociated cell cultures of the hippocampus and cerebellum of fetal rats. Addition of 1 ng/ml EGF enhanced the survival of cultured neurons of both brain regions. Both K-252a (10-200 nM) and staurosporine (1-100 nM) blocked the survival-promoting effects of EGF in a concentration-dependent manner. These results suggest that activation of protein kinase(s) is involved in the neurotrophic effects of EGF.

Topics: Alkaloids; Animals; Brain; Carbazoles; Cell Survival; Cells, Cultured; Epidermal Growth Factor; Indole Alkaloids; Neurons; Protein Kinase C; Rats; Recombinant Proteins; Staurosporine

The ras gene product (p21) is thought to transduce signals from various growth and differentiation factors. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various factors. Nerve growth factors (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as 20% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal growth factor (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic fibroblast growth factor, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).

Topics: Animals; Carbazoles; Epidermal Growth Factor; GTP-Binding Proteins; Guanosine Triphosphate; In Vitro Techniques; Indole Alkaloids; Nerve Growth Factors; PC12 Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Rats; Signal Transduction

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.

Topics: Animals; Carbazoles; Epidermal Growth Factor; Genistein; Guanosine Diphosphate; Guanosine Triphosphate; Indole Alkaloids; Isoflavones; Kinetics; Nerve Growth Factors; PC12 Cells; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Tyrosine

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.

Topics: Adenosine; Adrenal Gland Neoplasms; Animals; Carbazoles; Cyclic AMP; Enzyme Activation; Epidermal Growth Factor; Indole Alkaloids; Microtubule-Associated Proteins; Nerve Growth Factors; Oligopeptides; Pheochromocytoma; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured