Abstract
Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.
Highlights
Nerve growth factor (NGF)1 and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems [1]
The finding that cAMP can stimulate CREB-binding protein (CBP) activity is in accord with a previous study demonstrating that overexpression of protein kinases (PKAs) in the cells enhanced the transcriptional activity of CBP [9]
Previous studies revealed that NGF can regulate cyclic AMP response element binding (CREB) activity by inducing phosphorylation of CREB and interaction with CBP [6, 9]
Summary
Materials—2.5sNGF, basic FGF, IL-6, TNF-␣, IFN-␥, all-trans retinoic acid, and dibutyryl adenosine 3Ј,5Ј-cyclic monophosphate (Bt2cAMP) were from Sigma. Horse serum, and fetal calf serum were from Life Technologies, Inc. Plasmid DNAs—The following Gal4-CBP chimera constructs have been described previously: Rc/RSV Gal4-(1–147), pGal-CBP-(1– 460), pGal-CBP-(227– 460), pGal-CBP-(721–1679), pGal-CBP-(1678 –2441), pGal-CBP-(1678 –1843), pGal-CBP-(1844 –1956), pGal-CBP-(1961– 2039), pGal-CBP-(2060 –2179), pGal-CBP-(2173–2288), pGal-CBP(2288 –2441), and pGal-CBP(full-length) [21]. The expression vectors for the constitutively active MAPKK mutant (SS/DD) and the dominant-negative p42/44MAPK mutant (T192A) were obtained from Dr J. The expression plasmid containing the constitutive active subunit of PKC was a kind gift from Dr M. PKA-deficient PC12 cells [25], which were a kind gift from Dr J. Transient transfections of PC12cells and ND7 cells were performed using the calcium phosphate procedure as described by Gorman [26]. For each transfection a -galactosidase expression plasmid was used as an internal control.
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