Early Changes in the Synthesis of Nuclear and Cytoplasmic Proteins Are Induced by Nerve Growth Factor in Differentiating Rat PC12 Cells

  • Eric M. Shoote
  • Published 2002


Differentiation of rat pheochromocytoma PC12 cells into neuron-like cells was induced by nerve growth factor (NGF) and changes in the apparent rate of synthesis of cellular proteins were analyzed. Attention was particularly focused on the first few hours of exposure to NGF before significant neurite outgrowth was detectable. Cultures were pulse-labeled for 1-h periods with [35S]methionine and proteins were extracted from various subcellular fractions and analyzed by one-dimensional gradient and two-dimensional equilibrium and nonequilibrium gel electrophoresis. The results showed that although the general level of protein synthesis remained constant, by 8 h NGF increased the apparent rate of synthesis of '~11 cytoplasmic and 5 nuclear proteins. For several of these proteins, the effect was apparently NGF-specific, since no induction was observed in dibutyryl cAMP-treated cells. Of interest was the following observation: of the five nuclear proteins, NGF increased the synthesis of two proteins with Mrs of 56,000 [doublet] and 50,000 D that were associated with a biochemically and morphologically defined nuclear matrix fraction. A cytoplasmic protein, with an Mr of 92,000 D (pI 4.8) appeared to be induced de novo by NGE NGF also decreased the rate of synthesis of several cytoplasmic and nuclear proteins of low molecular mass (<40,000 D). Since only 1-h pulses of [35S]methionine were used, and since experiments with actinomycin D showed that most of these NGF-induced early changes in rates of synthesis included a transcription-dependent step, it seems likely that early effects of NGF include activation of specific genes. T HE PC12 clonal cell line derived from a transplantable rat pheochromocytoma (15) exhibits several characteristics of adrenal medullary chromaffin cells and responds to nerve growth factor (NGF) ~ by expressing many of the phenotypic properties of sympathetic neurons, including neurite outgrowth (15, 16, 57). Although the molecular mechanism by which NGF acts in PC12 cells as well as in normal neurons remains largely unknown, pharmacological data and in vitro transcription assays suggested that several parameters of the NGF-induced differentiation of PC12 cells involve transcriptional events, i.e., induction of neurite outgrowth (3), increased synthesis of NGF-inducible large external glycoprotein (43), and increases in ornithine decarboxylase (OrnDCase) mRNA (9), in fos mRNA (14), and in acetylcholinesterase activity (17). In addition, ongoing translation is necessary for the neurite outgrowth response of PC12 cells (16; Tiercy, J.-M., and E. M. Shooter, unpublished data) as well as of other cell types, such as chick sensory neurons (46). 1. Abbreviations used in this paper: dbcAMP, dibutyryl cAMP; NEPHGE, nonequilibrium pH gradient gel electrophoresis; NGF, nerve growth factor; OrnDCase, ornithine decarboxylase; RSB, buffer containing 10 mM NaCI, 10 mM Tris-HCl, pH 7.4, and 1.5 mM MgCI2; TNM sucrose, buffer containing 25 mM NaCI, 5 mM MgC12, 10 mM triethanolamine, pH 7.4, and 250 mM sucrose. However, a number of studies on PC12 cells, using oneand two-dimensional gel electrophoresis techniques, have led to the conclusion that NGF-stimulated neurite outgrowth does not involve qualitative changes in the synthesis and accumulation of the most abundant cellular proteins. Several quantitative changes have been described, including increased levels of the NGF-inducible large external protein (43), an 80-kD protein (44), a 55-56-kD protein (11), the neurofilament proteins and vimentin (36), a 25-kD glycoprotein immunologically cross-reactive with Thy-l.1 antigen (53), and the tau (7) and MAP1 proteins (7, 18), the latter two occurring concommitantly with increases in microtubule mass and neurite extension (7). The rate of synthesis of a significant fraction of total cellular proteins was found to be modulated similarly by NGF and by two different cAMP analogs, the major cytoskeletal proteins remaining unaffected (12). NGF also markedly increased the release into the medium of a 70-kD and a 30-kD protein (11). The synthesis of a 34-kD single-strand DNA binding protein was inhibited by NGF (2). Among the early effects of NGF are transient synthesis of proto-oncogenefos proteins (5, 31), an increase in OrnDCase (9, 22) and tyrosine hydroxylase (19) activities, and stimulation of the phosphorylation of a variety of proteins (18, 23, 34, 62). Although the PC12 protein phosphorylation studies were © The Rockefeller University Press, 0021-9525/86/12/2367/12 $1.00 The Journal of Cell Biology, Volume 103 (No. 6, Pt. 1), Dec. 1986 2367-2378 2367 performed at very early times, i.e., within minutes after addition of NGE none of the analyses described above considered possible NGF-induced changes in rates of protein synthesis during the first few hours of NGF treatment, nor did they examine the subcellular localization of the induced proteins, except for the Thy-l-cross-reactive glycoprotein (57). It should also be pointed out that most of the previous studies on NGF-induced proteins describe changes in steady-state amounts, because of the use of the cumulative labeling method. In this paper, we present a study on changes in rates of protein synthesis during NGFand dibutyrl cAMP (dbcAMP)-induced differentiation of PC12 cells, focusing attention on the first 8 h of NGF treatment. Oneand two-dimensional gel electrophoretic analyses of [35S]methionine pulse-labeled proteins extracted from PC12 cultures (+NGF) after subcellular fractionation showed that the rates of synthesis of several cytoplasmic and nuclear proteins were increased within the first few hours after NGF treatment and that the time courses of these increases were not all the same. Subnuclear fractionation studies furthermore revealed that two of these NGF-induced proteins were preferentially associated with the nuclear matrix fraction. Finally, the increased resolution of two-dimensional gel electrophoresis allowed us to detect the NGF-induced de novo synthesis of one cytoplasmic protein with an apparent Mr of 92,000 D and a pI of 4.8. We also present evidence that most of these early changes in rates of synthesis include a transcriptiondependent step and that induction of several of these proteins was not observed in cultures treated with dbcAMP. Materials and Methods

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@inproceedings{Shoote2002EarlyCI, title={Early Changes in the Synthesis of Nuclear and Cytoplasmic Proteins Are Induced by Nerve Growth Factor in Differentiating Rat PC12 Cells}, author={Eric M. Shoote}, year={2002} }