Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation Ingrid M. Nijholt a, 1 , Amalia M. Dolga a, 1 , Anghelus Ostroveanu a , Paul G.M. Luiten a , Martina Schmidt b,2 , Ulrich L.M. Eisel a, ⁎ ,2 a Department of Molecular Neurobiology, University of Groningen, 9750 AA, Haren, The Netherlands b Department of Molecular Pharmacology, University of Groningen, 9713 AD, Groningen, The Netherlands ABSTRACT ARTICLE INFO Article history: Received 1 April 2008 Accepted 11 May 2008 Available online 16 May 2008 Keywords: A-kinase anchoring protein Cyclic adenosine monophosphate Protein kinase B/Akt cAMP-dependent protein kinase Exchange proteins directly activated by cAMP Neuron AKAP150 HT-4 In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Cyclic adenosine monophosphate (cAMP) is one of the most common and versatile intracellular signaling compounds and has been implicated in the downstream transfer of molecular messages upon stimulation by a large number of hormones, neurotransmitters, prostaglandins and odorants. It functions as a universal and highly modulated second messenger for events underlying a wide variety of cellular processes such as central metabolic events, cardiac and smooth muscle contraction, secretory processes, ion channel con- ductance, learning and memory, cell growth and differentiation, apoptosis and inflammatory responses [1]. Although originally cAMP- dependent protein kinase (PKA) was thought to be the major if not the sole effector of cAMP, meanwhile other targets have been identified, in particular, exchange factors directly activated by cAMP (Epac) proteins. To date, there are two variants of Epac known, Epac1 and Epac2, and both were initially characterized as exchange factors for the small GTPases Rap1 and Rap2 [2]. Epac1 is ubiquitously distributed with predominant expression in the thyroid, kidney, ovary, skeletal muscle, and specific brain regions, whereas Epac2 is mainly expressed in the brain and adrenal gland [3,4]. The available information on the functional role of Epac in neurons is still rather limited. It was shown that Epac enhances neurotransmitter release in glutamatergic synapses of the rat brain calyx of Held [5] and in the crayfish neuromuscular junction [6]. In dorsal root ganglion neurons, Epac mediates the translocation and activation of protein kinase C (PKC) leading to the establishment of inflammatory pain [7] and in cerebellar granule cells Epac can modulate neuronal excitability [8]. Since PKA and Epac are co-expressed in many tissues, an increase in intracellular cAMP levels can lead to the activation of both cAMP targets, thus specificity and coordination of cAMP signaling is urgently required. Members of the A-kinase anchoring protein (AKAP) family were shown to play a pivotal role in the intracellular targeting and compartmentalization of cAMP signaling pathways [9,10]. AKAPs represent a group of more than 50 identified functionally related proteins. Although they share a few primary structure similarities, they all have the ability to bind the PKA regulatory subunit [11]. Besides a binding site for PKA many AKAPs contain distinct binding Cellular Signalling 20 (2008) 1715–1724 ⁎ Corresponding author. Department of Molecular Neurobiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. Tel.: +31 50 3632366; fax: +31 50 3632331. E-mail addresses: i.m.nijholt@rug.nl (I.M. Nijholt), a.m.dolga@rug.nl (A.M. Dolga), a.ostroveanu@rug.nl (A. Ostroveanu), p.g.m.luiten@rug.nl (P.G.M. Luiten), m.schmidt@rug.nl (M. Schmidt), u.l.m.eisel@rug.nl (U.L.M. Eisel). 1 These authors contributed equally to the work. 2 These authors share the senior authorship. 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.05.001 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig