The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain

Abstract

A-kinase anchoring proteins (AKAPs) influence the spatial and temporal regulation of cAMP signaling events. Anchoring of PKA in proximity to certain adenylyl cyclase (AC) isoforms is thought to enhance the phosphorylation dependent termination of cAMP synthesis. Using a combination of immunoprecipitation and enzymological approaches, we show that the plasma membrane targeted anchoring protein AKAP9/Yotiao displays unique specificity for interaction and the regulation of a variety of AC isoforms. Yotiao inhibits AC 2 and 3, but has no effect on AC 1 or 9, serving purely as a scaffold for these latter isoforms. Thus, Yotiao represents an inhibitor of AC2. The N terminus of AC2 (AC2-NT), which binds directly to amino acids 808 -957 of Yotiao, mediates this interaction. Additionally, AC2-NT and Yotiao (808 -957) are able to effectively inhibit the association of AC2 with Yotiao and, thus, reverse the inhibition of AC2 by Yotiao in membranes. Finally, disruption of Yotiao-AC interactions gives rise to a 40% increase in brain AC activity, indicating that this anchoring protein functions to directly regulate cAMP production in the brain. 2). This family of proteins functions to target PKA and other cAMP effector proteins to specific regions of the cell allowing for increased signaling specificity. Several members of this diverse protein family bind other receptors, channels, or enzymes to form tightly regulated signaling modules, facilitating PKA interactions with its downstream effectors. In addition, these signaling modules often constitute feedback loops between kinases and phosphatases or recruit phosphodiesterases to terminate the cAMP signal (3).Our labs previously identified a complex containing AKAP79/ 150 and type 5 adenylyl cyclase (AC), that facilitates the spatiotemporal organization of cAMP signaling (4). AKAP79 inhibits AC5 activity, while providing a mechanism for feedback inhibition via PKA phosphorylation of anchored AC5. However, with Ϸ30 gene families of AKAPs identified thus far, and nine mammalian isoforms of AC, it begs the question, does AKAP scaffolding of AC and PKA represent a general paradigm for cAMP signaling? The AKAP Yotiao represents an ideal test case to begin to address this question. Yotiao is a splice variant of the AKAP9 gene and is present on the plasma membrane (5, 6). In addition to PKA, Yotiao binds protein phosphatase 1 (PP1), NMDA receptors, the heart potassium channel subunit KCNQ1, and the IP 3 R1 (5, 7-9). Interestingly, AKAP anchored PKA phosphorylation of the NMDA receptor, KCNQ1, and IP 3 R1 is necessary for each effector's normal function (8-11). For example, disruption of the KCNQ1 association with Yotiao gives rise to long QT syndrome, a type of heart arrhythmia that can be fatal (12). The requirement for a tight coupling between PKA and KCNQ1, and other effectors suggests that cAMP production must also be tightly regulated, perhaps as even part of this complex.We now show that Yotiao is associated with brain and heart adenylyl cyclases. Yotiao displays specificity am...

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

“…AKAP5 is known to directly interact with AC5 and AC6 and scaffold them to AMPA receptors (39), and AKAP5-bound RII causes phosphorylation and inhibition of these enzymes (40). Furthermore, AKAP9/Yotiao directly inhibits AC2 and AC3 (41). Therefore, AKAP5 may bring AC into close proximity with GPR30 for the receptor complex to inhibit cAMP production.…”

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells

“…Such a proposal is quite speculative at present because it is not known whether such complexes exist or how these domains of cAMP are maintained. However, evidence is accumulating that ACs interact with a variety of proteins, such as A-kinase-anchoring proteins, protein phosphatase 2A and the actin cytoskeleton (Bauman et al, 2006;Crossthwaite et al, 2006;Head et al, 2006;Piggott et al, 2008). Additionally, the assembly of signalling complexes is often accompanied by their residence in lipid rafts (Pike, 2003).…”

“…Experimental evidence is accumulating for the existence of cAMP microdomains in which cAMP targets are discretely and selectively regulated in a manner that bears little relation to changes in gross cellular levels of cAMP (Bacskai et al, 1993;Hempel et al, 1996;Nikolaev et al, 2006;Rich et al, 2001;Zaccolo and Pozzan, 2002). Recent studies suggest that adenylyl cyclases (ACs), the originators of cAMP, can actually orchestrate their own microenvironment by recruiting a variety of signalling and scaffolding molecules (Bauman et al, 2006;Chou et al, 2004;Crossthwaite et al, 2006;Dupre et al, 2007;Piggott et al, 2008). Thus, the nine differently regulated isoforms of AC (Sunahara and Taussig, 2002) can also contribute significantly to the diversity of cellular cAMP microdomains.…”

AKAP9 Is Essential for Spermatogenesis and Sertoli Cell Maturation in Mice

“…One major AKAP family member, AKAP9, was first described as an N-methyl-D-aspartic acid (NMDA) receptor anchor in the brain (Lin et al 1998) and was further characterized in the brain and heart (Feliciello et al 1999;Kurokawa et al 2004;Ciampi et al 2005;Chen et al 2007;Piggott et al 2008;Chen and Kass 2011;Chopra and Knollmann 2011). Mutations in human AKAP9 have been implicated in breast cancer (Milne et al 2011), sporadic papillary thyroid carcinomas (Lee et al 2006), and long QT syndrome (Chen et al 2007).…”