N-methyl-d-aspartate signaling to nuclear activator protein-1 through mechanisms different from those for kainate acid signaling in murine brain

doi:10.1016/S0306-4522(98)00647-2

Neuroscience

Volume 90, Issue 2, 22 February 1999, Pages 519-533

https://doi.org/10.1016/S0306-4522(98)00647-2 Get rights and content

Abstract

Protein de novo synthesis is mainly under the control at the level of gene transcription by transcription factors in cell nuclei in eukaryotes. The systemic administration of N-methyl-d-aspartate resulted in selective but transient potentiation of binding of a radiolabeled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 in murine hippocampus, without markedly affecting binding of probes for other transcription factors. By contrast, kainate induced more potent and more persistent potentiation of activator protein-1 binding in the hippocampus than N-methyl-d-aspartate. The protein synthesis inhibitor cycloheximide was effective in significantly preventing the potentiation by N-methyl-d-aspartate, but not that by kainic acid at the doses used. Moreover, kainic acid induced much more and longer expression of immunoreactive c-Fos protein in the hippocampus than N-methyl-d-aspartate. However, neither N-methyl-d-aspartate nor kainic acid induced expression of cyclic AMP response element binding protein phosphorylated at serine133 in the hippocampus from 10 min to 24 h after the administration. Instead, kainate was more potent than N-methyl-d-aspartate in facilitating both dephosphorylation at serine and phosphorylation at tyrosine of particular nuclear proteins in the hippocampus.

These results suggest that N-methyl-d-aspartate and kainate signals may be differentially transduced into cell nuclei to express the activator protein-1 complex through molecular mechanisms which differ from phosphorylation of cyclic AMP response element binding protein at serine133 but involve serine dephosphorylation and/or tyrosine phosphorylation of particular nuclear proteins in the murine hippocampus.

Section snippets

Materials

[α-³²P]Deoxy-ATP (111 TBq/mmol) was purchased from NEN/DuPont (Boston, MA, U.S.A.). Klenow fragment of DNA polymerase I was supplied by Takara Biochemicals (Kyoto, Japan). Nick columns were obtained from Pharmacia LKB (Uppsala, Sweden). Oligonucleotides with different base sequences were all provided by Inter Tech Co. (Tokyo, Japan). Rabbit polyclonal antibodies against CREB peptide (123–136) and CREB peptide phosphorylated at serine133 (pCREB) were purchased from Upstate Biotechnology Inc.

Experimental conditions

Preincubation with poly(dI-dC) was invariably necessary to minimize nonspecific binding of radiolabeled probes in nuclear extracts as much as possible. Under the routine conditions, binding of radiolabeled probes for AP1, c-Myc and Zif/268 was competed with the respective unlabeled probes at a concentration range of two to 200 times higher molar ratio, but not with those having a point mutation at the individual consensus elements at the same concentration range (data not shown). Removal of NaF

Phosphatase inhibitors

From the results, there is no doubt that particular nuclear protein phosphatases at least in part participate in mechanisms underlying the modulation of DNA binding activities of transcription factors with zinc-finger motifs, in addition to those with leucine-zipper motifs as demonstrated previously.[38] In particular, removal of both NaF and GP markedly potentiated binding of probes for YY1 and GREBd in nuclear extracts kept at 2°C. This means that DNA binding activities of these transcription

Conclusions

It thus appears that systemic NMDA and kainate signals differentially modulate de novo synthesis of the individual target proteins at the level of gene transcription through the AP1 complex consisting of the c-Fos protein expressed by mechanisms which differ from phosphorylation of CREB at serine133 but involve protein tyrosine phosphorylation and/or serine dephosphorylation in cell nuclei.

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research to Y.Y. from the Ministry of Education, Science, Sports and Culture, Japan.

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Transcriptional regulation is one of the most important functions of polyamines in the nucleus of eukaryotic cells. The addition of the endogenous polyamines spermine and spermidine markedly increased DNA binding activity of the transcription factor activator protein-1 (AP1) in a concentration-dependent manner at a concentration range of 50 to 500 μM in nuclear extracts of murine whole brain when determined in the absence of added MgCl₂ on gel retardation electrophoresis. Similar but less potent potentiation was seen with DNA binding of cAMP responsive element binding protein (CREB), while both polyamines were ineffective in affecting that of c-Myc irrespective of the addition of MgCl₂. Unlabeled AP1 probe was invariably more potent in competing for AP1 binding than unlabeled CREB probe in either the presence or absence of spermine and spermidine. In addition to whole brain, both polyamines significantly increased AP1 binding in retina, adrenal and pituitary, without significantly affecting that in spleen. Moreover, ultraviolet and circular dichroism spectra analyses revealed that these two polyamines induced DNA topological transition of AP1 probe under the conditions favorable for the increase in AP1 binding. These results suggest that both spermine and spermidine may modulate gene transcription through cis- and trans-actions on AP1 binding in the nucleus in murine central and peripheral structures with high excitability.

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¹: Present address: Department of Pharmacology, Osaka Dental University, 8-1 Kuzuha Hanazono-cho, Hirakata, Osaka 573-1121, Japan

View full text

N-methyl-d-aspartate signaling to nuclear activator protein-1 through mechanisms different from those for kainate acid signaling in murine brain

Abstract

Section snippets

Materials

Experimental conditions

Phosphatase inhibitors

Conclusions

Acknowledgements

Neurochem. Int.

Neurochem. Int.

Cell

Neurosci. Lett.

J. biol. Chem.

Neurosci. Lett.

J. biol. Chem.

Cell

Brain Res.

Brain Res.

Analyt. Biochem.

Neurochem. Int.

Neuroscience

Neurosci. Lett.

Brain Res.

Stimulation of protein tyrosine phosphorylation by NMDA receptor activation

Science

Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation

Nature

Mouse brain c-fos mRNA distribution following a single electroconvulsive shock

J. Neurochem.

Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide

Eur. J. Biochem.

Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyrus

Nature