Dephosphorylation of pCREB by protein serine/threonine phosphatases is involved in inactivation of Aanat gene transcription in rat pineal gland

Abstract

The rat pineal gland is a suitable model to investigate neurotransmitter-controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N-acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE-stimulation for 8 h induced accumulation of PSP1-catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A-CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland. Keywords: arylalkylamine N-acetyltransferase, ICER, norepinephrine, pCREB dephosphorylation, protein serine/threonine phosphatases, rat pineal gland. In many neuronal and neuroendocrine cells, neurotransmitter-driven signal transduction cascades converge on the activation of transcription factors that control cellular activity by initiating or repressing transcription of their target genes. The rat pineal gland represents an excellent model to study the regulatory mechanisms acting on the cAMP-controlled transcription factors CREB [cAMP responsive element (CRE)-binding protein] and ICER (inducible cAMP early repressor) and to link them with a defined output, the biosynthesis of the hormone melatonin (for review see Korf et al. 1998;Stehle et al. 2001).As in other systems, CREB is constitutively expressed in the pineal gland and its transcriptional impact depends on phosphorylation at serine 133, but not on de novo protein synthesis (Maronde et al. 1999a). Phosphorylation of CREB is induced by norepinephrine (NE; Roseboom and Klein 1995;Tamotsu et al. 1995;Korf et al. 1996), which is released exclusively during the night from sympathetic nerve endings and activates cAMP-dependent protein kinase A (PKA) type II via a b-adrenergic/cAMP mechanism (Korf et al. 1998;Maronde et al. 1999b). Phosphorylated (p) CREB enhances transcription of genes bearing a CRE in their promoter regions. Most importantly, NE elicits a more than 150-fold rise in the transcription of the gene encoding the arylalkylamine N-acetyltransferase (AANAT), the ratelimiting enzyme in melatonin biosynthesis (Borjigin et...

Diurnal variation in CREB phosphorylation and PER1 protein levels in lactotroph cells of melatonin-proficient C3H and melatonin-deficient C57BL mice: similarities and differences

“…Immunocytochemical demonstration of CREB phosphorylated at Ser133 (pCREB) has been used as a marker for the activity state of the lactotroph cells. CREB phosphorylation is known to play an important role in the rhythmic activation and synchronization of gene expression in the central circadian rhythm generator, the SCN (Ginty et al 1993;Gau et al 2002), and in peripheral oscillators, such as the pineal organ (Maronde et al 1999;von Gall et al 2000;Koch et al 2003) and the pars tuberalis (McNulty et al 1996;von Gall et al 2002). Moreover, several studies have provided evidence that a cyclic AMP-protein kinase ApCREB cascade is involved in the control of Prl gene expression (Keech et al 1992;Liang et al 1992;Coleman et al 1996;Hayakawa et al 2002) and that photoperiodic regulation of the lactotroph axis depends at least in part upon changes in Prl mRNA levels (Stirland et al 2001;Johnston et al 2003).…”

Os efeitos do treinamento físico aeróbio sobre a síntese de melatonina pineal em ratos.

“…A presença da CREB fosforilada é muito importante também para a manutenção da atividade da AANAT, pois, quando a CREB é desfosforilada, se tem uma rápida queda na atividade da enzima (KLEIN; ROSEBOOM; KOCH et al, 2003;TAMOTSU et al, 1995).…”

Avaliação das alterações causadas pelo câncer sobre a produção de melatonina na glândula pineal.

“…A presença da CREB fosforilada é muito importante também para a manutenção da atividade da AA-NAT, pois, quando a CREB é desfosforilada, se tem uma rápida queda na atividade da enzima Koch et al, 2003;Roseboom, Klein, 1995;Tamotsu et al, 1995).…”