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

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.